Cell lines

ABSTRACT

There is provided inter alia a process for stabilizing a eukaryotic cell line which expresses PylRS and tRNAPyl and which is suitable for incorporation of a gene encoding a target protein containing one or more non-natural amino acids encoded by a nonsense codon which comprises culturing said cell line under conditions in which the adverse effect of tRNAPyl expression on cell viability and/or cell growth is reduced or eliminated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/430,542, filed Mar. 23, 2015, which is the U.S. national phaseapplication of International Patent Application No. PCT/EP2013/069887,filed Sep. 24, 2013, which claims priority to U.S. ProvisionalApplication Nos. 61/705,116, filed Sep. 24, 2012, and 61/862,495, filedAug. 5, 2013, the entire disclosures of each of which are incorporatedby reference herein in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file entitled 304_0039USD1_SeqListing.txt and having a size of 168kilobytes filed with the application is incorporated herein by referencein its entirety.

FIELD OF THE INVENTION

This invention relates to stable eukaryotic cell lines suitable for usein incorporating non natural amino acids into proteins and to processesfor preparing them. This invention also relates to proteins withincorporated non natural amino acids which are suitable for conjugationwith other proteins, with drugs or other moieties e.g. to allow halflife extension and to corresponding protein conjugates. Further, theinvention relates to novel amino acid derivatives.

BACKGROUND TO THE INVENTION

The site-specific introduction of non natural amino acids (nnAAs) into atarget protein provides a significant advantage for the generation offunctionalized protein conjugates over non specific methods (Wang etal., 2011). A variety of non natural amino acids are available thatcontain moieties that provide bioorthogonal sites for conjugationchemistry and enable specific reactions to occur at these sites. Controlover the positions of the conjugation site enables products with optimalfunction by avoiding active sites and essential protein functionaldomains. In addition, this allows for the generation of a homogeneousand predictably modified product that improves the functionalcharacteristics and purification of the product.

Site specific incorporation of nnAAs in bacterial cells has beenachieved through amino acid substitution approaches and through theengineering of orthogonal aminoacyl tRNA synthetases that charge onlytheir cognate tRNAs with a non natural amino acid. The position of thenon natural amino acid in the target protein can be specified by avariety of codons within the gene sequence, but most often it isdirected to amber codons. The variety of proteins that can be expressedin E. coli and other prokaryotic based systems, however, is limited bythe protein folding machinery of these organisms. Eukaryotic expressionsystems (such as mammalian expression systems) are capable of expressinga wider variety of proteins including those that require glycosylationfor optimal therapeutic function (e.g. G-CSF, insulin, epoietin alpha)exist as protein complexes (e.g. antibodies), or requireposttranslational modifications such as disulfide bond formation (e.g.atrial natriuretic factor) that are not accessible in bacterial systems.

Systems for the introduction of nnAAs into mammalian cells have beendeveloped either through transfection of in vitro charged tRNAs (Hechtet al., 1978; Kohrer et al., 2001; Kohrer et al., 2003) or geneticallyencoded using orthogonal aminoacyl tRNA synthetase/tRNA pairs (Mukai etal 2008, Liu W. et Al., 2007; Wang W., 2007; Ye, S. et Al., 2008;Sakamoto, K et Al., 2002; Takimoto, J. et Al., 2009; Chen, P. et Al.,2009). Chemically acylated tRNAs are not reacylated and thus their useis prohibitive to large scale protein synthesis whether in vitro or invivo. Genomically encoded RS/tRNA pairs are required to be orthogonal tothe host cell in order to retain the specificity of nnAA insertion.

In use of orthogonal aminoacyl tRNA synthetase/tRNA pairs, orthogonalityof the RS and tRNA is achieved through mutations at key sites to enablespecificity for a nnAA while at the same time reducing or eliminatingrecognition of canonical amino acids, and host tRNAs. The tRNA may alsobe modified to prevent cognition by host RSs and to recognize amber stopcodons. Several RS/tRNA pairs have been developed including the E. coliTyrRS/B. stearothermophilus tRNAtyr (Liu, W., 2007; Ye et al., 2008;Sakamoto et al., 2002) and E. coli TyrRS/E. coli tRNAtyr (Wang, W.,2007; Takimoto et al., 2009).

It has been observed that one orthogonal RS/tRNA pair naturally evolvedin a subset of archaebacteria (methanogenic archaea bacteria thatcatabolize methylamines) which has specificity for the amino acidpyrrolysine. Pyrrolysine uses a 21st aminoacyl-tRNA synthetase,naturally evolved to be orthogonal to all other amino acids and tRNAs.

Pyrrolysine is a natural amino acid, the only one that is authenticallyspecified by an amber codon. Blight et al., 2004 showed that PylRS andtRNApyl can incorporate Pyrrolysine at amber codons in E. coli. Theyalso showed that the wild type (“WT”) PyLRS is naturally promiscuous andcan incorporate analogs of Lysine.

Yokoyama et al (EP1911840) demonstrated that the PylRS/tRNA system isorthogonal in eukaryotic cells and showed the incorporation of severalnnAAs into a target proteins encoded by amber codons in bacterial cells.These authors also identified key amino acid residues in pylRS that formthe amino acid binding pocket and function in selecting pyrrolysine overother canonical amino acids. Mutations at this site generated mutantsable the recognize and aminoacylate the tRNApy with AzZ-lys (Yanagisawa2008)

This orthogonality extends to bacteria and eukaryotic cells.

PylRS is a naturally promiscuous synthetase that has naturally evolvedto exclude lysine, but will incorporate lysine analogs without mutationincluding azides, alkynes and alkenes, (Yanagisawa et al, 2008; Neumannet al. 2008; Mukai et al., 2008; Nguyen et al., 2009). The basis of thisspecificity is dependent on hydrophobic interactions between amino acidresidues of the pylRS binding pocket with the pyrrol ring of pyrrolysinethat stabilizes and correctly positions the amino acid in the activesite of the synthetase (Kavran et al., 2007). This RS/tRNA pair has beenintroduced via transient transfection into bacterial, yeast andmammalian cells and shown to be effective for incorporation of a numberof non-natural amino acids into target proteins.

For instance, EP 1911840 demonstrates incorporation of N-ε-boc-Lysineinto a target protein in E. coli cells.

The expression of tRNA in eukaryotic cells requires two internalpromoters within the tRNA coding sequence. The consensus sequences ofsuch promoters are known as the A-Box and B-Box (Naykova et al., 2003).

Although certain prokaryotic-derived tRNAs naturally carry sequencesthat function as an internal promoter and can be expressed in animalcells without modifications, or with changes that generate an intragenicpromoter sequence but do not alter the function of the tRNA or itsrecognition by its cognate RS, tRNAPyl does not contain such promoter.Furthermore, the D loop where A-Box and B-Box are normally present isunusually small and the introduction of said A-Box and B-Box woulddestroy its function as reported in yeast by Hancock et al (2010) andconfirmed by the inventors in mammalian cells.

WO2007099854 describes the use of a eukaryotic snRNA promoter to drivetRNAPyl expression in eukaryotic cells. DNA constructs described thereincomprise the tRNApyl gene, a transcription terminator sequence placed 3′of said tRNA gene, and a promoter sequence that induces transcription byRNA Polymerase II or III such as U1 snRNA promoter or U6 snRNA promoterplaced 5′ to said tRNApyl gene.

Mammalian expression is of particular interest as it allows for theproduction of fully folded proteins and protein complexes like fulllength antibodies that are challenging or currently inaccessible toprokaryotic systems or yeast cells.

Transient transfection experiments of genes encoding the pyrrolysineaminoacyl tRNA synthetase (pylRS) and its tRNApyl, in both human(HEK293) and hamster (CHO) cells, have shown that the pylRS/tRNA pairefficiently incorporates nnAAs into a target protein at sites designatedby an amber stop codon in mammalian cells (see for instance Mukai 2008).

EP1911840 teaches the introduction of a vector carrying a WT PylRS, avector carrying a tRNApyl gene, and a vector carrying a target genewhere an amber mutation is introduced at the site where the lysinederivative is to be inserted. The only technique utilized to introducethe vectors is transient transfection. In fact, nowhere in the patentapplication the selection of stable clones is mentioned nor appliedexperimentally.

WO09038195 describes the generation of mutant PylRS enzymes in order toimprove its catalytic activity, and allow incorporation of non naturalamino acids derived from lysine with bulky side chain structures.

In particular, WO09038195 describes a mutation at position 384 (referredto methanosarcina mazei PylRS amino acid sequence) whereby Tyr384 isreplaced with Phenylalanine, among other amino acids. It is hypothesizedthat due to the fact that Tyr384 interacts with a substrate amino acid,particularly with its main chain (Kavran 2007, Nozawa 2009) there islikelihood that the enzyme catalytic activity would be enhancedindependently of the substrate.

As noted above, expression based on the PylRS and tRNApyl orthogonalpair has hitherto only been achieved in transiently stable eukaryoticcell lines. Transiently stable cell lines are not suitable for thereliable manufacture of commercial products; indeed the presentinventors believe that the biologic products on the market today derivedfrom mammalian cells are exclusively generated by stable cell lines.

Therefore there remains a need in the art to develop methods for theproduction of stable eukaryotic cells containing the PylRS and tRNApylorthogonal pair thereby to facilitate production of proteins containingnnAAs on a commercial scale.

The present invention addresses the aforementioned need.

Pyrrolysine analogs, defined as amino acid derivatives recognized byeither native or genetically evolved PylRS and incorporated intoproteins at amber codon sites, have been disclosed in the past few yearsand reviewed, for instance, by Feckner et. al (Fekner, Li, & Chan, 2010)and Liu et al. Analogs bearing functional groups or post translationalmodifications have been site-specifically incorporated into proteinsusing pylRS-tRNApyl systems. Several studies, see e.g. Yanagisawa et al,focused on mutations within the PylRS enzyme in order to accommodateanalogs in which the N6 substituent were an aromatic ring within thebinding pocket pyrrolysine. Others, for instance Nguyen et al (also inWO2010/139948), and Li et al (also in WO2011/044255) focused onidentification of pyrrolysine analogs which do not carry a bulky N6substituent, with the result of obtaining simpler analogs which would besimple to synthesize and interact with native pylRS/tRNApyl pairs. Thereremains a need to develop further pyrrolysine analogs. Whilstpyrrolysine analogs made thus far have been restricted to those evolvedfrom a lysine backbone, the present inventors have generated pyrrolysineanalogs successfully incorporated into proteins with nativepylRS/tRNApyl pairs starting from a variety of amino acid structures.

Antibody drug conjugates (ADCs), composed of recombinant chimeric,humanized or human antibodies covalently bound by means of syntheticlinkers to highly cytotoxic drugs, have been developed in recent yearsin order to target cytotoxic drugs to tumor cells. The right combinationof antibodies targeting tumor associated antigens, a potent toxins andappropriate conjugation chemistry can be very effective at deliveringthe toxin directly to the tumor cells, while avoiding toxicity of thedrug to normal tissue.

ADCs developed so far are heterogeneous mixtures of conjugated andunconjugated antibody, depending on the chemistry of the conjugationused when generating the ADC. In particular, the random nature of themost commonly used conjugation protocols results in a collection ofspecies with varying numbers of drugs conjugated per antibody molecule(DAR) as well as varying conjugation sites. Common conjugationchemistries include lysine side-chain based conjugation, which resultsin a wide range of species due to the large availability of lysineresidues in a typical antibody. More site-specific conjugations havebeen obtained through engineering of cysteine residues to producereactive thiol groups, resulting in nearly homogeneous ADCs.

Her2 tumor associated antigen, a member of the EGFR family, has beensuccessfully targeted in breast cancer with Herceptin, an anti-Her 2antibody, however, the antibody itself is effective in a limited groupof patients. A more potent form, ado-trastuzumab emtansine, which has atoxin linked to it, is now available. Ado-trastuzumab emtansine is ableto effectively treat patients who are refractory to Herceptin, due tothe ability of ado-trastuzumab emtansine to deliver a toxin to thecytoplasm of the cancer cell. The conjugation chemistry being used byado-trastuzumab emtansine and Brentuximab vedotin, exploits existingcysteine residues that normally form disulfide bonds, and more recently,engineered free cysteine residues. This approach has led to theproduction of heterogeneous mixtures of ADC with different numbers ofdrug at different positions on the mAb. The linkers used includethioether (Kadcyla) as well as dipeptide linkers (Adcetris), the latterbeing specifically cleaved by lysosomal acid hydrolases. Both types oflinkers appear to be effective, but not optimal. Conventional Cys or Lysdirected bioconjugation methods such as those used for manufacture of byado-trastuzumab emtansine, Brentuximab vedotin, and gemtuzumabozogamicin permit premature release of toxin prior to tumor cellengagement. Gemtuzumab ozogamicin, which was approved in 2000, waswithdrawn from the market in 2010, due to high toxicity due to the useof an acid labile linker which caused intolerable release of the toxinfrom the ADC in the blood.

Therefore there remains a need in the art to develop highly homogeneousADCs, where the conjugation sites and the number of drugs per antibodyare well controlled.

The present inventors have found that through use of site specificincorporation of nnAAs and subsequent conjugation of antibodies at thesite of nnAA it is possible to generate homogeneous and potent ADCs.Furthermore, the present inventors have found sites, within the IgGconstant region, which can be used for conjugation without disruptingthe specificity of the binding of the antibody or its pharmacokineticproperties in vivo.

SUMMARY OF THE INVENTION

According to the present invention there is provided a process forpreparing a stable eukaryotic cell line which expresses pylRS andtRNApyl and which is suitable for incorporation of a gene permittingexpression of a target protein containing one or more non-natural aminoacids encoded by an amber codon.

The invention is derived from the inventors' findings concerning thesource of instability of cell lines prepared by prior art methods.

It may be observed that conventional eukaryotic cells such as CHO, andHEK293 cells that express pylRS and tRNApyl without any gene permittingexpression of a target protein containing one or more non-natural aminoacids encoded by an amber codon adopt a phenotype indicative of cellulartoxicity, including higher proportion of dead cells in the culture, arounded cellular morphology, loose attachment of the cells to the growthplates, and decreased cell growth rates. The inventors observed thatupon subsequent expression of said gene permitting expression of atarget protein the health of cells appeared to improve noticeably. Itappears to the inventors that the toxicity is associated with theexpression in the system of high amounts of the tRNApyl which in theabsence of nnAA induces the extension of essential) housekeeping genesthat terminate in an amber stop codon (Liebman and Sherman 1976; Liebmanet al., 1976).

Thus, without being bound by theory, toxic effects of tRNApyl may be aconsequence of imperfect orthogonality occurring when high levels oftRNApyl are present in the absence of a target protein.

While the tRNApyl is for the most part orthogonal in mammalian cells, itis possible that the tRNApyl may be inefficiently aminoacylated by oneof the host RSs in the absence of nnAA (where the natural enzyme, pylRSis vacant). In cells expressing high levels of the tRNA a significantamount of aminoacylated tRNA may be generated which forces irregularamber suppression of essential genes.

Accordingly the inventors have surmised that stable cell lines may beproduced by processes which have as their objective a reduction in ormasking of the apparently toxic effects of tRNApyl.

As a first aspect of the invention, therefore, there is provided aprocess for stabilizing a eukaryotic cell line (particularly a mammaliancell line) which expresses PylRS and tRNAPyl and which is suitable forincorporation of a gene encoding a target protein containing one or morenon-natural amino acids encoded by an amber codon which comprisesculturing said cell line under conditions in which the adverse effect oftRNAPyl expression on cell viability and/or cell growth is reduced oreliminated.

As a second aspect of the invention, there are provided decoy aminoacids (dnnAAs) of formula VII as described herein, which have the meritof reducing or masking the toxic effects of tRNApyl when added to theculture media during the production or maintenance of said stable celllines.

According to a third aspect of the invention, there are provided novelnon natural amino acids analogs of pyrrolysine of Formulae V and VI asdescribed herein, which have the merit of being straightforward toprepare, in being readily incorporated into proteins (typically withoutloss of bioactivity when used appropriately) and in providing usefulmeans for bioconjugation.

According to a fourth aspect of the invention, there are providedmethods to obtain highly homogeneous and active Antibody Drug Conjugatesthrough site specific insertion of non natural amino acids atpre-determined positions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Scheme detailing the iterative introduction of RS/tRNA elementsand selection steps performed for the development of a platform cellline and subsequent expression cell line.

FIG. 2. Functional comparison of parental and sorted cells isolatedduring the development of a platform cell line.

FIG. 3. Characterization of expressed anti-IL-6 IgG K274 containing alys-azide nnAA from a stable cell line.

FIG. 4. Illustration of how tRNA is the limiting component to the systemand background activity has a cytostatic effect.

FIG. 5. Non-Reducing SDS-PAGE gel of PEGylation of an anti-IL-6 AzAbwith a linear 20kPEGAlkyne (A) and Non-Reducing SDS-PAGE gel ofPEGylation of Anti-IL-6-LysAzide274h with 20KPEG cyclooctyne (B).

FIG. 6. Non-Reducing SDS-PAGE gel of Bispecific Conjugation of anAnti-IL-6-LysAzide274h to 31A12-20KPEG alkyne (A) and Reducing SDS-PAGEgel of Bispecific Conjugation of an Anti-IL-6-LysAzide274h to31A12-20KPEG alkyne (B).

FIG. 7. Evidence that FGF21 containing a propargyl-lysine nnAA atposition R131 is efficiently PEGylated and retains function in vivo.

FIG. 8. Evidence that toxin conjugated antibodies and antibody fragmentsdemonstrate specific activity in vitro.

FIG. 9. Non Reducing gel of anti-Her2 Antibody containing an azidereacting with 20K PEG Cyclo-Alkyne (A) and Reducing gel of anti-Her2Antibody containing an azide reacting with 20K PEG Cyclo-ALKYNE (B)

FIG. 10. Non-reducing SDS-PAGE gel of 20KPEGylation ofAnti-Her2-LysAzide274h70l (A) and Reducing SDS-PAGE gel of 20KPEGylation of 4D5 AzAb-4 (B)

FIG. 11. Reducing gel of anti-Her2 Antibody containing an azide reactingwith 20K PEG terminal alkyne in the presence of Copper (A) and NonReducing gel of V anti-Her2 Antibody containing an azide reacting with20K PEG terminal alkyne in the presence of Copper (B)

FIG. 12. Reducing gel of anti-PSMA scFv incorporating Lys-Azideconjugated to 20K linear PEG cyclooctyne (A) and Reducing gel ofanti-PSMA scFv with nnAA Lys-Azide conjugated to MMAF-VCP-cyclooctyne(B)

FIG. 13. In vitro functional assays examine the function and specificityof decoy nnAA competition with lys azide.

FIG. 14. In vitro functional assays examine the efficacy of dnnAAsfunction in competition with lys azide and their effect on backgroundamber suppression in cells containing pylRS/tRNA.

FIG. 15. Growth rate and viability assay of pylRS/tRNA containing cellsgrown in the presence or absence of decoy nnAA.

FIG. 16. Population analyses of pylRS/tRNA function in cells treatedwith decoy nnAA.

FIG. 17. Growth rates of cells containing the pylRS/tRNA pair examinedin cultures treated with decoy nnAA.

FIG. 18. PEGylation of azide containing monoclonal antibodies. Lane 1:Untreated Antibody, Lane 2: Antibody with pyrrolysine analog Formula V.1incorporated into heavy chain and subjected to PEGylation conditions;Lane 3: Antibody with pyrrolysine analog Formula VI.1 incorporated intoheavy chain and subjected to PEGylation conditions.

FIG. 19. HIC chromatogram of a 4D5-Auristatin F antibody drug conjugatewith the antibody originally containing the pyrrolysine analog FormulaVI. 1, incorporated into the heavy chain.

FIG. 20. SDS PAGE analysis of PEGylated 4D5 positional mutants

FIG. 21. Reaction analysis of Auristatin conjugation to 4D5-AzAb's withthe azide incorporated at different positions by HIC chromatography.

FIG. 22. SDS-PAGE of ADCs derived from positional mutants of 4D5 azide

FIG. 23. Potency and selectivity assessment of positional variants of4D5-2AzAb Auristatin antibody drug conjugates by an in vitrocytotoxicity assay versus high and low express Her2 cell lines.

FIG. 24. Reaction analysis of 4D5-2AzAb(HC274) conjugation to afluorescent dye by HIC chromatography.

FIG. 25. Pharmacokinetics and stability of 4D5 modified at position H274

FIG. 26. Overlay of HIC chromatograms of unconjugated antibody and4D5-2AzAb(HC274)-Auristatin F antibody drug conjugate prepared withAuristatin Cyclooctyne derivative.

FIG. 27. In vitro antitumor activity of 4D5-2AzAb (HC274)-AF conjugateagainst Her2 positive tumor cell lines

FIG. 28. In vivo antitumor activity of 4D5-2AzAb (HC274)-AF. Tumorprogression expressed as the mean tumor size for each group (A) or thepercent survival (B).

FIG. 29. SDS PAGE analysis of 4D5-2AzAb/FGF21 bispecific. Lane 1: MWMarkers, Lane 2: FGF21 untreated, Lane 3: 4D5-FGF21 bispecific reaction,Lane 4: 4D5-2AzAb.

FIG. 30. ELISA assay scheme and data for the detection of a bi-specificantibody constructed with a full length mAb containing a nnAA atposition H274 (anti-Her2) and a scFv directed against IL6. A) ELISAshowing capture of the full length mAb (4D5) and detection of thebispecific using IL6. B) ELISA showing functional binding of the fulllength mAb (4D5) to the extracellular domain of Her 2. and subsequentdetection of the mAb. C) ELISA assay showing functional binding of themAb to the Her2 extracellular domain and scFv binding to IL6.

FIG. 31. SDS PAGE analysis of 4D5 AzAb-FGF21 bispecific

FIG. 32. SDS-PAGE analysis of reaction mixture of 20 kDa PEGylation of4D5-2AzAb(HC274) under CuAAC conditions and TBTA

FIG. 33. SDS PAGE analysis of the product of a 20kPEGylation to 4D5 AzAbwith CuAAC and THPTA

FIG. 34. A, B, PAGE analysis of 2 kDa PEGylation of 4D5-2AzAb (HC274)with CuAAC/THPTA under reducing and non reducing conditions. C, HICchromatogram of the final reaction mixture of 2 kDa PEGylation of4D5-AzAb

FIG. 35. In vitro cytotoxic effect of DAR4 4D5-AF ADC's

FIG. 36. In vitro cytotoxic effect of 4D5-Amanitin ADC's

FIG. 37. In vitro cytotoxic effect of 4D5-AF ADC's obtained via CUAACand SPAAC chemistries.

FIG. 38. HIC chromatogram of the reaction mixture of a 4D5-2AzAb(HC274)conjugation to a Auristatin F derivative under CuAAC conditions.

FIG. 39. Gel Mobility assay of site-specifically modified Herceptin AzAbheavy chain with 20KDa PEG alkyne.

FIG. 40. Analysis of Herceptin AzAb conjugated to AuristatinF-cyclooctyne. A) HIC analyses of the untreated Herceptin-AzAb and theproduct of the conjugation reaction. B) Gel mobility assays of untreatedand conjugation reactions by SDS-PAGE under reducing (B) andnon-reducing conditions (C).

FIG. 41. Analysis of Herceptin AzAb conjugated to Auristatin F-alkyne.A) HIC analyses of the untreated Herceptin-AzAb and the product of theconjugation reaction. B) Gel mobility assays of untreated andconjugation reactions by SDS-PAGE under reducing (B) and non-reducingconditions (C).

FIG. 42. In vitro cytotoxicity of Herceptin ADCs

FIG. 43. A schematic showing a non-cleavable linker with a functionalhandle (Y) for attaching to the antibody at one terminus, a spacer whichbridges the two components of the antibody drug conjugate (ADC) andprovides the functional groups necessary to attach to the antibody andto the drug. and a complimentary functional group (X) for coupling tothe drug.

FIG. 44. A schematic showing a linker with a functional handle (Y) thatincludes an alkyne group.

FIG. 45. A schematic showing a non-cleavable linker with a functionalhandle (Y) at the antibody attachment site that includes a vinyl halide.

FIG. 46. A schematic showing a non-cleavable linker with a functionalhandle (Y) at the antibody attachment site that includes a reactivearomatic ring substituted with a silyl group and either a halide ortriflate, tosylate or mesylate at the LG position.

FIG. 47. A schematic showing a non-cleavable linker with a functionalhandle (Y) at the antibody attachment site that includes a reactiveazide group at the terminus.

FIG. 48. A schematic showing a cleavable linker for an antibody drugconjugate (ADC) that includes a cleavage site.

FIG. 49. A schematic showing a cleavable linker with a functional handle(Y) at the antibody attachment site that includes an alkyne.

FIG. 50. A schematic showing a cleavable linker in which the cleavagesite and spacer are reversed in order with the cleavage site at the drugattachment site.

FIG. 51. A schematic showing a cleavable linker with a functional handle(Y) at the antibody attachment site that includes a vinyl halide.

FIG. 52. A schematic showing a cleavable linker with a functional handle(Y) at the antibody attachment site that includes a reactive aromaticring substituted with a silyl group and either a halide or triflate,tosylate or mesylate at the LG position.

FIG. 53. A schematic showing a cleavable linker with a functional handle(Y) at the antibody attachment site that includes a reactive azide groupat the terminus.

FIG. 54. A schematic showing a linker that includes a cycloalkyne at oneterminus for attachment to the antibody via azidealkyne cycloaddition; acarbon chain spacer attached to the cycloalkyne which is then attachedto a valinecitrulline peptide; wherein the C-terminus of citrulline iscoupled to a p-amino-benzoyl carbamate (PABC) which is connected to theN-terminus of monomethyl auristatin F (MMAF).

FIG. 55. A schematic showing PEGylation of anti-IL-6 antibody with NNAALys-Azide incorporated at position 274 of heavy chain with 20KPEGterminal alkyne (Anti-IL-6-LysAzide274h).

FIG. 56. A schematic showing PEGylation of anti-IL-6 antibody with NNAALys-Azide incorporated at position 274 of heavy chain(Anti-IL-6-LysAzide274h) with 20KPEG CYCLOOCTYNE(bicyclo[6.1.0]non-4-yne-linked PEG).

FIG. 57. A schematic showing conjugation of an anti-IL-6 (antibody)-antiIL-23 (scFv-PEG) bispecific antibody.

FIG. 58. A schematic showing conjugation of anti-Her2 Antibody havingnnAA lys-azide incorporated at position 274 of heavy chain(Anti-Her2-LysAzide274h) with monomethyl auristatin F (MMAF)-cyclooctynederivative.

FIG. 59. A schematic showing PEGylation with 20K Linear PEG-cyclooctyneto anti-Her2 Antibody (Anti-Her2-LysAzide274h).

FIG. 60. A schematic showing PEGylation with 20KPEG alkyne to anti-Her2Antibody (Anti-Her2-LysAzide274h).

FIG. 61. A schematic showing conjugation of anti PSMA scFv with NNAAsubstituted at position 117 (anti-PSMAscFV-117) with monomethylauristatin F(MMAF)-valine-citruline-p-amino-benzoyl-carbonate-cyclooctynederivative.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID No 1: PylRS Methanosarcina mazei WT amino acid sequence

SEQ ID No 2: PylRS Methanosarcina mazei Y384F mutant amino acid sequence

SEQ ID No 3: PylRS Methanosarcina barkeri WT amino acid sequence

SEQ ID No 4: PylRS Desulfitobacterium hafniense amino acid sequence

SEQ ID No 5: PylRS Methanosarcina acetivoran amino acid sequence

SEQ ID No 6: PylRS Methanococcoides burtonii amino acid sequence

SEQ ID No 7: PylRS Methanosarcina thermophila amino acid sequence

SEQ ID No 8: PylRS Methanosalsum zhilinae amino acid sequence

SEQ ID No 9: PylRS Methanohalobium evestigatum amino acid sequence

SEQ ID No 10: PylRS Methanohalophilus mahii amino acid sequence

SEQ ID No 11: PylRS Desulfotomaculum gibsoniae amino acid sequence

SEQ ID No 12: PylRS Desulfosporosinus meridiei amino acid sequence

SEQ ID No 13: PylRS Desulfotomaculum acetoxidans amino acid sequence

SEQ ID No 14: PylRS Methanosarcina mazei WT nucleotide sequence

SEQ ID No 15: PylRS Methanosarcina mazei Y384F mutant nucleotidesequence

SEQ ID No 16: PylRS Codon optimized Methanosarcina mazei nucleotidesequence

SEQ ID No 17: PylRS Methanosarcina barkeri nucleotide sequence

SEQ ID No 18: PylRS Desulfitobacterium hafniense nucleotide sequence

SEQ ID No 19: PylRS Methanosarcina acetivorans nucleotide sequence

SEQ ID No 20: PylRS Methanococcoides burtonii nucleotide sequence

SEQ ID No 21: PylRS Methanosarcina thermophila nucleotide sequence

SEQ ID No 22: PylRS Methanosalum zhilinae nucleotide sequence

SEQ ID No 23: PylRS Methanohalobium evestigatum nucleotide sequence

SEQ ID No 24: PylRS Desulfotomaculum gibsoniae nucleotide sequence

SEQ ID No 25: PylRS Methanohalophilus mahii nucleotide sequence

SEQ ID No 26: tRNApyl Methanosarcina barkeri

SEQ ID No 27: tRNApyl Methanosarcina acetivorans

SEQ ID No 28: tRNApyl Methanosarcina mazei

SEQ ID No 29: tRNApyl Methanococcoides burtonii

SEQ ID No 30: tRNApyl Desulfobacterium hafniense

SEQ ID No 31: H1/TO Promoter

SEQ ID No 32: U6 snRNA Promoter

SEQ ID No 33: SNR52 Promoter

SEQ ID No 34: H1 Promoter

SEQ ID No 35: U6-tRNApyl construct

SEQ ID No 36: GFP nucleotide sequence

SEQ ID No 37: GFP amino acid sequence

SEQ ID No 38: GFPY40 Amino acid Sequence

SEQ ID No 39: anti-IL-6 (28D2) Gamma Nucleotide Sequence

SEQ ID No 40: anti-IL-6 (28D2) Gamma Amino acid Sequence

SEQ ID No 41: anti-IL-6 (28D2) Gamma_amber K274 Nucleotide Sequence

SEQ ID No 42: anti-IL-6 (28D2) Gamma_amber K274 Amino acid Sequence

SEQ ID No 43: anti-IL-6 (28D2) Kappa Nucleotide Sequence

SEQ ID No 44: anti-IL-6 (28D2) Kappa Amino acid Sequence

SEQ ID No 45: anti-Her2 (4D5) gamma Nucleotide sequence

SEQ ID No 46: anti-Her2 (4D5) gamma amino acid sequence

SEQ ID No 47: anti-Her2 (4D5) gamma_K274amber nucleotide sequence

SEQ ID No 48: anti-Her2 (4D5) gamma_K274amber amino acid sequence

SEQ ID No 49: anti-Her2 (4D5) gamma_T359amber nucleotide sequence

SEQ ID No 50: anti-Her2 (4D5) gamma_T359amber amino acid sequence

SEQ ID No 51: anti-Her2 (4D5) Kappa nucleotide sequence

SEQ ID No 52: anti-Her2 (4D5) Kappa amino acid sequence

SEQ ID No 53: anti-Her2 (4D5) Kappa D70 amber nucleotide sequence

SEQ ID No 54: anti-Her2 (0.4D5) Kappa D70 amber amino acid sequence

SEQ ID No 55: anti-Her2 (0.4D5) Kappa E81 amber nucleotide sequence

SEQ ID No 56: anti-Her2 (4D5) Kappa E81 amber amino acid sequence

SEQ ID No 57: anti-PSMA scFv nucleotide sequence

SEQ ID No 58: anti-PSMA scFv amino acid sequence

SEQ ID No 59: anti-PSMA scFv_117amber nucleotide sequence

SEQ ID No 60: anti-PSMA scFv_117amber amino acid sequence

SEQ ID No 61: FGF21 WT nucleotide sequence

SEQ ID No 62: FGF21 WT amino acid sequence

SEQ ID No 63: FGF21 R131amber nucleotide sequence

SEQ ID No 64: FGF21 R131amber amino acid sequence

SEQ ID No 65: FGF21 F12amber nucleotide sequence

SEQ ID No 66: FGF21 F12amber amino acid sequence

SEQ ID No 67: FGF21 L60amber nucleotide sequence

SEQ ID No 68: FGF21 L60amber amino acid sequence

SEQ ID No 69: FGF21P90amber nucleotide sequence

SEQ ID No 70: FGF21P90amber amino acid sequence

SEQ ID No 71: FGF21 P140amber nucleotide sequence

SEQ ID No 72: FGF21 P140amber amino acid sequence

SEQ ID No 73: GFPY40 nucleotide Sequence

SEQ ID No 74: Herceptin nucleotide sequence Heavy Chain

SEQ ID No 75: Herceptin amino acid sequence Heavy Chain

SEQ ID No 76: Herceptin H274 Nucleotide sequence Heavy Chain

SEQ ID No 77: Herceptin H274 amino acid sequence Heavy Chain

SEQ ID No 78: Herceptin nucleotide sequence Light Chain

SEQ ID No 79: Herceptin amino acid sequence Light Chain

SEQ ID No 80: 3× Flag tag sequence

SEQ ID No 81: 5×Pro-6×His tag

SEQ ID No 82: portion of human IgG1 Heavy Chain (constant region)

SEQ ID No 83: portion of SEQ ID No 52 (framework region)

SEQ ID No 84: portion of SEQ ID No 52 (framework region)

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “alkyl” refers to an aliphatic linkage or substituent,typically containing 1-6 e.g. 1-4 carbon atoms and can be straight chainor branched. Examples include methyl, ethyl, n-propyl, i-propyl, n-butyland t-butyl.

The term “alkenyl” refers to an aliphatic linkage or substituent,typically containing 2-6 e.g. 2-4 carbon atoms and can be straight chainor branched and which is unsaturated in respect of containing at leastone C═C moiety. A specific example is a terminal alkene group in whichthe C═C moiety is at the terminus. Examples of alkenyl include ethenyl,propen-1-yl, propen-2-yl, and 2-methyl-propen-2-yl.

The term “alkyne” or “alkynyl” refers to an aliphatic linkage orsubstituent, typically containing 2-6 e.g. 2-4 carbon atoms and can bestraight chain or branched and which is unsaturated in respect ofcontaining at least one C≡C moiety. A specific example is a terminalalkyne group in which the C≡C moiety is at the terminus. Examples ofalkynyl groups include —C≡CH and —C═C—CH₃.

The term “aryl” refers to an aromatic ring structure that can be part ofa linkage or part of a substituent. Aryl moieties may contain one ring(e.g. phenyl) or two rings (e.g. naphthyl) or more than two rings,provided that at least one ring is aromatic. Aryl groups may besubstituted e.g. by one or more (e.g. one or two, such as one)substituent selected from alkyl, alkenyl, alkynyl, fluoroalkyl, halogen,alkoxy, nitro and cyano. An exemplary aryl is phenyl.

The term “heteroaryl” refers to a heteroaromatic ring structure that canbe part of a linkage or part of a substituent. The heteroaromatic ringmay contain 1-4 (more usually 1-3 e.g. one or two) heteroatoms selectedfrom O, N and S. Heteroaryl moieties may contain one ring or two ringsor more than two rings, provided that at least one ring isheteroaromatic. Example groups containing one 6 membered ring includepyridine and pyrimidine. Example groups containing one 5 membered ringinclude pyrrole, furan, thiophene, oxazole, thiazole, diazole,thiadiazole and tetrazole. Heteroaryl moieties that contain two ringsmay contain heteroatoms in one or both rings. Examples include quinolineand isoquinoline. Heteroaryl groups may be substituted e.g. by one ormore (e.g. one or two, such as one) substituent selected from alkyl,alkenyl, alkynyl, fluoroalkyl, halogen, alkoxy, nitro and cyano.

The term “methyl” or “Me” refers to a CH₃ group

The term “OMe” refers to a O—CH₃ group.

The term “ethyl” or “Et” refers to a CH₂CH₃ group.

The term “OEt” refers to a O—CH₂CH₃ group

The term “tBu” refers to a C(CH₃)₃ group

The term “OtBu” refers to a O—C(CH₃)₃ group.

The term “OBn” or “OBenzyl” refers to a O—CH₂-Ph group

The term “OFmoc” or “OCH₂Fluorene” refers to the following structure:

The term “Phenyl” or “Ph” refers to a benzene ring as in the followingstructure:

The term “allyl” refers to a CH₂—CH═CH₂ group

The term “ethyl chloride” refers to a CH₂CH₂—Cl group

The term “azide” and “azido” refers to a N═N(+)=N(−) or N₃ functionalgroup.

The term “azidoalkyl” means alkyl substituted by azido, especiallyterminal azido. Examples include —(CH₂)_(n)N₃ wherein n=1-4.

The term “haloalkyl” means alkyl substituted by one or more (e.g. 1, 2or 3, especially 1 or 2 such as 1) halogen atoms (eg Cl or F atoms).Examples include —CF₃ and —CH₂CH₂Cl.

The term “propargyl” refers to a methyl group appended to a terminalalkyne. It is denoted by —CH₂—C≡C—H.

The term “amide” refers to a —C(═O)—NH— linkage.

The term “carbamate” refers to a —O—C(═O)—NH— linkage.

The term “ester” refers to a —C—C(═O)—O—C linkage

The term “alkoxy” refers to the group —O-alkyl.

The term “ketone” refers to a C—C(═O)—C linkage.

The term “pyrrolysine analog” means an amino acid derivative recognizedby either native or genetically evolved PylRS and incorporated intoproteins at a nonsense codon site.

The term “the side chain of one of the 20 natural amino acids” refers tothe group R in the formula HOOC—CHR—NH₂ relating to the 20 natural aminoacids known by their single letter codes A, C, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W and Y. Either L or S stereochemistry (or amixture thereof) is intended, although L stereochemistry is preferred.

The term “cell viability” refers to a determination of living (viable)or dead cells, based on a total cell sample, within the context of cellscultured in vitro. A cell is considered viable if it has the ability togrow and develop. Viability assays are based on either the physicalproperties of viable cells such as membrane integrity or on theirmetabolic activity.

The term “cell growth” refers to cellular proliferation as measured bythe number of cell divisions over a period of time. Growth is measuredby tracking the cell density (cell/mL) of a culture over time.

The term “stable expression” refers to the expression of a protein whichis achieved by integration of the gene (or corresponding cDNA) ofinterest into the target cell's chromosome.

The term “stable integration” therefore refers to the integration of thegene (or corresponding cDNA) of interest into the target cell'schromosome: Initially the gene of interest has to be introduced into thecell, subsequently into the nucleus and finally it has to be integratedinto chromosomal DNA. Stably transfected cells can be selected andcultured in various ways: for instance, a selection marker isco-expressed on either the same or on a second, co-transfected vector. Avariety of systems for selecting transfected cells exists, includingresistance to antibiotics such as neomycin phosphotransferase,conferring resistance to G418, dihydrofolate reductase (DHFR), orglutamine synthetase. The culture of the transfected cells can be doneeither in bulk to obtain a mixed population of resistant cells, or viasingle cell culture, to obtain cell clones from one single integrationevent.

The term “target gene” refers to the gene encoding for the protein to bemodified via insertion of nnAAs.

Various Embodiments According to the Invention

“Decoy Amino Acid” Approach

According to an embodiment, there is provided a process wherein theconditions in which the adverse effect of tRNApyl on cell viabilityand/or cell growth is reduced or eliminated include conditions in whichthere is present in the medium in which the cell line is cultured adecoy amino acid which is a substrate for PylRS (i.e. is aminoacylatedand is loaded onto the tRNApyl) but which is incapable of beingincorporated into an extending protein chain.

Thus, a process to incorporate non natural amino acids into a proteinmay include the following steps:

-   -   1. Introduce a decoy amino acid into the growth medium of cells        that is readily recognized and activated by the orthogonal RS        for the cognate tRNA    -   2. Introduce the RS and tRNA into a eukaryotic cell, on one or        more plasmids    -   3. Select for cells containing the RS and tRNA expression        cassettes    -   4. Isolate one or more stable clones expressing the RS protein        and tRNA thereby generating a platform cell line. Cells capable        of non natural amino acid incorporation rates of greater than        30%, for example greater than 40% or 50% or 60% or 70% or 80% or        preferably greater than 90% are selected.    -   5. Introduce the cDNA of the target protein whereby one or more        amber codons has been introduced at the position or positions        into which the non natural amino acid is to be incorporated    -   6. Isolate a stable clone expressing high levels (greater than        0.5-10 pg/cell/day) of the target gene product    -   7. Grow the cell line in the absence of the decoy amino acid,        but in the presence of the non natural amino acids allowing for        incorporation at the amber codon.

According to this embodiment, when the decoy amino acid is present inthe cell-line containing the PylRS and tRNA synthetase pair, it binds tothe PylRS and is aminoacylated to the tRNA. It is then passed on to theribosome, where the tRNA binds the amber codon, but the acylatedamino-blocked decoy is disabled from forming a peptide bond, thus theprotein terminates at this site.

In one embodiment, the decoy is not present once the cDNA of the targetprotein is introduced in the cell.

Alternatively, the decoy amino acid is maintained in the culture mediumwhen the cDNA encoding the target protein is introduced into the cell.

In an alternative embodiment, expression of the target protein occurs inthe presence of the decoy amino acid. Thus according to this embodiment,the decoy amino acid and a desired non-natural amino acid which ispreferentially used by the PylRS are both added to (or present in) thefermentation medium during target protein production.

A decoy amino acid should not be added to or present in the fermentationmedium if it competes with the desired non-natural amino acid forbinding to the PylRS to any significant extent.

In another embodiment, expression of the target protein does not occurin the presence of the decoy amino acid (e.g. following elimination fromthe fermentation medium). Thus according to this embodiment, the decoyamino acid is not introduced into the fermentation media during theexpression of the target protein. Only the desired non-natural aminoacid which preferentially binds the PylRS is added to (or present in)the fermentation during target protein production. According to thisembodiment, the decoy amino acid is utilized in the culture mediumthroughout the selection and isolation of platform cell lines containingthe pylRS/tRNA. After introduction and selection of a target genecontaining one or more amber codons, the decoy amino acid is removed.

A plurality of (e.g. 2, 3, 4, 5, 6 or 7 or more) decoy amino acids maybe employed if desired.

A further aspect of the invention is a process for production of astable eukaryotic cell line which is capable of expressing PylRS andtRNAPyl and which is suitable for incorporation of a gene encoding atarget protein containing one or more non-natural amino acids encoded byan nonsense codon which comprises (a) in one or more steps introducinginto a eukaryotic cell line genes encoding PylRS and tRNAPyl and suchthat PylRS and tRNAPyl are stably expressed in said cell line (b)culturing or selecting the resultant cell line in the presence of adecoy amino acid which is a substrate for PylRS but which is incapableof incorporation into an extending protein chain thereby to reduce theadverse effect of tRNAPyl on cell viability and/or cell growth.

A further aspect of the invention is a process for production of astable eukaryotic cell line which is capable of expressing PylRS,tRNAPyl and a target protein containing one or more non-natural aminoacids encoded by a nonsense codon which comprises (a) in one or moresteps introducing into a eukaryotic cell line genes encoding PylRS andtRNAPyl such that PylRS and tRNAPyl are stably expressed in said cellline (b) culturing or selecting the resultant cell line in the presenceof a decoy amino acid which is a substrate for PylRS but which isincapable of incorporation into an extending protein chain thereby toreduce the adverse effect of tRNAPyl on cell viability and/or cellgrowth (c) introducing into said eukaryotic cell line a gene encoding atarget protein containing one or more non-natural amino acids such thatsaid target protein is stably expressed in said cell line and (d)expressing the target protein in the absence of said decoy amino acid.

A further aspect of the invention is a process for production of astable eukaryotic cell line according to any one of the aforementionedaspects wherein the culturing or selection of the cell line is performedin the presence of a decoy amino acid which is a substrate for PylRS butwhich is incapable of incorporation into an extending protein chainthereby reducing the adverse effect of tRNAPyl on cell viability and/orcell growth.

“Target First” Approach

According to an alternative embodiment, there is provided a processwherein the conditions in which the adverse effect of tRNAPyl on cellviability and/or cell growth is reduced or eliminated include conditionsin which a target protein containing one or more non-natural amino acidsencoded by a nonsense codon is also expressed by said cell line.

Thus, a process to incorporate non natural amino acids into a proteinmay include the following steps:

-   -   1. Introduce the target gene containing an amber codon at a        position into which the non natural amino acid is to be        incorporated into a eukaryotic cell on one or more plasmids    -   2. Isolate a pool of cells or clone that expressed the target        protein at high levels (greater than 0.5 or greater than 10        pg/cell/day)    -   3. Introduce the RS and tRNA into these cells, on one or more        plasmids, and select for clones containing the RS and tRNA    -   4. Grow the cell line in the presence of the non natural amino        acids allowing for incorporation at the amber codon and isolate        cells which show an incorporation efficiency of the non natural        amino acid at the desired sites of greater than 30%, for example        greater than 40% or 50% or 60% or 70% or 80% or preferably        greater than 90%.

A further embodiment is a stable eukaryotic cell line which expressesPylRS and tRNAPyl and also expresses under the control of an induciblepromoter a decoy protein containing one or more non-natural amino acidsencoded by an amber codon.

A further aspect of the invention is a process for production of astable eukaryotic cell line which expresses PylRS, tRNAPyl and a targetprotein containing one or more non-natural amino acids encoded by anamber codon which comprises (a) introducing into a eukaryotic cell linea gene encoding a target protein containing one or more non-naturalamino acids encoded by an nonsense codon such that said gene is stablyintegrated in said cell line (b) in one or more steps furtherintroducing into said cell line genes encoding PylRS and tRNAPyl suchthat PylRS and tRNAPyl are stably expressed in said cell line and (c)culturing the resultant cell line in the presence of a source of the oneor more non-natural amino acids under conditions whereby tRNAPyl isexpressed only by said cell line at the same time as the target proteinis also expressed by said cell line thereby to reduce the adverse effectof tRNAPyl on cell viability and/or cell growth.

“Repressible tRNA” Approach

According to an alternative embodiment, there is provided a processwherein the conditions in which the adverse effect of tRNAPyl on cellviability and/or cell growth is reduced or eliminated include conditionsin which the expression of tRNAPyl occurs under the control of arepressible promoter.

There is also provided a eukaryotic cell line which expresses or iscapable of expressing PylRS and tRNAPyl in which expression of tRNAPyloccurs under the control of a repressible promoter.

Thus, a process to incorporate non natural amino acids into a proteinmay include the following steps:

-   -   1. Introduce the RS and tRNA, on one or more plasmids into a        eukaryotic cell, the latter containing a repressible promoter        element that enables control of tRNA expression.    -   2. Select for cells containing the RS and tRNA expression        cassettes under repressed conditions    -   3. Induce tRNA expression and isolate one or more stable clones        expressing high levels of the RS protein and tRNA or        demonstrating efficient suppression of amber codons in a        reporter gene thereby generating a platform cell line. Cells        capable of non natural amino acid incorporation rates of greater        than 30%, for example greater than 40% or 50% or 60% or 70% or        80% or preferably greater than 90% are selected.    -   4. Introduce the cDNA of the target protein whereby an amber        codon has been introduced at the position where the non natural        amino acid is to be incorporated    -   5. Isolate a stable clone expressing high levels (greater than        0.5-20 pg/cell/day) of the target gene product    -   6. Grow the cell line in presence of the non natural amino acids        allowing for incorporation at the amber codon.

According to this embodiment, high levels of expression of thesuppressor tRNA are avoided and amber suppression related cytotoxicityis prevented.

In such a process expression of the tRNA is suitably under the controlof repressible promoter such as the H1 and U6 promoters containingtetracycline responsive elements (TetO or TtA)(Herold 2008). A furtheraspect of the invention is a stabilized cell line which has beenprepared according to one of the aforementioned processes.

A further aspect of the invention is a process for production of astable eukaryotic cell line which is capable of expressing PylRS,tRNAPyl and which is suitable for incorporation of a gene encoding atarget protein containing one or more non-natural amino acids encoded byan nonsense codon which comprises (a) in one or more steps introducinginto a eukaryotic cell line genes encoding PylRS and tRNAPyl whichtRNAPyl is under the control of a repressible promoter and such thatPylRS and tRNAPyl are stably expressed in said cell line (b) culturingthe resultant cell line in the presence of a repressor such thatexpression of tRNAPyl is repressed thereby to reduce the adverse effectof tRNAPyl on cell viability and/or cell growth.

A further aspect of the invention is a process for production of astable eukaryotic cell line which is capable of expressing PylR, tRNAPyland a target protein containing one or more non-natural amino acidsencoded by an nonsense codon which comprises (a) in one or more stepsintroducing into a eukaryotic cell line genes encoding PylRS and tRNAPylwhich tRNAPyl is under the control of a repressible promoter and suchthat PylRS and tRNAPyl are stably expressed in said cell line (b)culturing the resultant cell line in the presence of a repressor suchthat expression of tRNAPyl is repressed thereby to reduce the adverseeffect of tRNAPyl on cell viability and/or cell growth (c) introducinginto said eukaryotic cell line a gene encoding a target proteincontaining one or more non-natural amino acids such that said targetprotein is stably expressed in said cell line and (d) expressing thetarget protein in the absence of said repressor such that tRNAPyl isexpressed.

“Decoy Protein” Approach

According to an alternative embodiment, there is provided a processwherein the conditions in which the adverse effect of tRNAPyl on cellviability and/or cell growth is reduced or eliminated include conditionsin which a decoy protein containing one or more non-natural amino acidsencoded by an amber codon is also expressed under the control of aninducible promoter by said cell line.

For example, the decoy protein is selected from: Green fluorescenceprotein, Red Fluorescence Protein, Yellow Fluorescence Protein, CyanFluorescence Protein, blue fluorescence protein, albumin, SEAP, Actin,b-2 microglobulin, glutathione-s-transferase, IgG, or a poly ambercontaining peptide.

Thus, a process to incorporate non natural amino acids into a proteinmay include the following steps:

-   -   1. Introduce a gene for a decoy protein containing an amber        codon into a eukaryotic cell which is under control of an        inducible promoter    -   2. Isolate a pool of cells or clone that contains the decoy        construct and upon induction is capable of expression of this        protein at high levels (greater than 0.1 or greater than 1        pg/cell/day)    -   3. Introduce the RS and tRNA into these cells, on one or more        plasmids, and select for clones containing the RS and tRNA    -   4. Isolate clones capable of incorporation efficiency of the non        natural amino acid at desired sites at rates greater than 30%,        for example greater than 40% or 50% or 60% or 70% or 80% or        preferably greater than 90% in the presence of the non natural        amino acid using the integrated decoy construct    -   5. Introduce the target gene containing an amber codon at a        position into which the non natural amino acid is to be        incorporated into a eukaryotic cell    -   6. Isolate clones capable of expression levels greater than 1        pg/cell/day of the target protein    -   7. Grow the cell line in the presence of the non natural amino        acids allowing for incorporation at the amber codon and isolate        cells which show an incorporation efficiency of the non natural        amino acid at the desired sites of greater than 30%, for example        greater than 40% or 50% or 60% or 70% or 80% or preferably        greater than 90%

A further aspect of the invention is a stable eukaryotic cell line whichexpresses pylRS, tRNApyl and a decoy protein under the control of aninducible promoter.

According to this embodiment, expression of the decoy protein can bediscontinued (e.g. by removal of the inducer for the promoter) when theexpression of target protein is commenced.

Suitable inducible promoters systems include conditionally activatedpromoters and promoter systems such as the tetracycline regulatedpromoters (TetO or tTA; TetOn and TetOFF), doxycycline-inducible (TRE)promoters, cAMP inducible promoters, glucocorticoid activated promotersystems, IPTG inducible promoters (lac), Cd2+ or Zn2+ induciblepromoters (methalloprotein promoters), interferon dependent promoters(e.g. murine MX promoter), HIV LTR promoters (Tat), DMSO induciblepromoters (GLVP/TAXI, ecdysone), and rapamycin inducible promoters(CID).

According to this embodiment, expression of the decoy protein can bediscontinued (e.g. by removal of the gene) when the expression of targetprotein is commenced using a recombination system.

Suitable systems include targeting recombination systems such as theCre/lox, the phi31C-based integration system, and Flp-FRT recombinationtechnology or by homologous recombination of the inserted cassettes.

A further aspect of the invention is a process for production of astable eukaryotic cell line which is capable of expressing PylRS,tRNAPyl and a decoy protein containing one or more non-natural aminoacids encoded by an nonsense codon and which is suitable forincorporation of a gene encoding a target protein containing one or morenon-natural amino acids encoded by a nonsense codon which comprises (a)in one or more steps introducing into a eukaryotic cell line genesencoding pylRS, tRNApyl and said decoy protein and such that PylRStRNAPyl and the decoy protein are stably expressed in said cell line (b)culturing the resultant cell line under conditions whereby tRNApyl isexpressed only by said cell line at the same time as the decoy proteinis also expressed by said cell line thereby to reduce the adverse effectof tRNApyl on cell viability and/or cell growth.

A further aspect of the invention is a process for production of astable eukaryotic cell line which is capable of expressing PylRS,tRNAPyl, a decoy protein and a target protein containing one or morenon-natural amino acids encoded by an nonsense codon which comprises (a)in one or more steps introducing into a eukaryotic cell line genesencoding pylRS, tRNApyl and a decoy protein said decoy protein beingexpressed under the control of an inducible promoter and such thatPylRS, tRNAPyl and the decoy protein are stably expressed in said cellline (b) culturing the resultant cell line under conditions wherebytRNApyl is expressed only by said cell line at the same time as thedecoy protein is also expressed by said cell line thereby to reduce theadverse effect of tRNApyl on cell viability and/or cell growth (c)introducing into said eukaryotic cell line a gene encoding a targetprotein containing one or more non-natural amino acids such that saidtarget protein is stably expressed in said cell line and (d) expressingthe target protein without expressing the decoy protein.

General

A further aspect of the invention is a stable eukaryotic cell lineobtained by or obtainable by any one of the aforementioned processes.

A further aspect of the invention is a stable eukaryotic cell lineobtained by or obtainable by a process according to a combination of twoor more of the aforementioned processes of modification of the system(i.e. use of decoy protein, decoy amino acid, repressiblepromoter/inducible promoter, introduction of the nonsense codoncontaining target gene prior to introduction of the Pyl-tRNA etc).

A further aspect of the invention is a process for preparing a targetprotein containing one or more non-natural amino acids encoded by annonsense codon which comprises culturing a stable eukaryotic cell lineas aforementioned in the presence of a source of the one or morenon-natural amino acids.

A further aspect of the invention is a process for preparing a targetprotein containing one or more non-natural amino acids encoded by anamber codon which comprises introducing into a stable eukaryotic cellline as aforesaid a gene encoding a target protein containing one ormore non-natural amino acids encoded by a nonsense codon such that thetarget protein is stably expressed in said cell line and expressing saidtarget protein in the presence of a source of the one or morenon-natural amino acids and in the absence of any inducer of expressionof the decoy protein.

A further aspect of the invention is a process for preparing a targetprotein containing one or more non-natural amino acids encoded by annonsense codon which comprises introducing into a stable eukaryotic cellline as aforesaid a gene encoding a target protein containing one ormore non-natural amino acids encoded by a nonsense codon such that thetarget protein is stably expressed in said cell line and expressing saidtarget protein in the presence of a source of one or more non-naturalamino acids and in the absence of any decoy amino acid.

A further aspect of the invention is a process for preparing a targetprotein containing one or more non-natural amino acids encoded by annonsense codon which comprises introducing into a stable eukaryotic cellline as aforesaid a gene encoding a target protein containing one ormore non-natural amino acids encoded by a nonsense codon such that thetarget protein is stably expressed in said cell line and expressing saidtarget protein in the presence of a source of the one or morenon-natural amino acids and in the absence of any repressor ofexpression of the tRNAPyl.

A further aspect of the invention is a process for preparing a targetprotein containing one or more non-natural amino acids encoded by annonsense codon which comprises introducing into a stable eukaryotic cellline as aforesaid a gene encoding a target protein containing one ormore non-natural amino acids encoded by a nonsense codon such that thetarget protein is stably expressed in said cell line and expressing saidtarget protein in the presence of a source of the one or morenon-natural amino acids.

A further aspect of the invention is process for preparing a chemicallymodified target protein which comprises preparing a target proteincontaining one or more non-natural amino acids encoded by a nonsensecodon which comprises introducing into a stable eukaryotic cell line asaforesaid a gene encoding a target protein containing one or morenon-natural amino acids encoded by a nonsense codon such that the targetprotein is stably expressed in said cell line, expressing said targetprotein in the presence of a source of the one or more non-natural aminoacids, and chemically modifying the resultant target protein.

Cell Lines for Use According to the Invention

The invention related to stable eukaryotic cell lines. Suitably the celllines are mammalian cell lines.

More preferably, the cell line is a CHO cell line, but also may be aHEK293, PERC6, COS-1, HeLa, VERO, or mouse hybridoma cell line. Furtherexamples are COS-7 and mouse myeloma cell lines.

CHO and HEK293 cells lines are particularly suitable.

Certain elements of the present invention may be used in the context ofa cell-free expression system wherein a synthesis reaction lysateobtained from a host cell comprises at least one component required forthe synthesis of polypeptides.

The synthesis reaction lysate may be obtained from bacterial oreukaryotic cells. Preferably, the synthesis reaction lysate is obtainedfrom eukaryotic cells, more preferably, from rabbit reticulocytes orwheat germ.

The cell-free expression system is capable of expressing WT PylRS andtRNApyl of the present invention, wherein tRNApyl is introduced into thecells used to obtain the synthesis reaction lysate with DNA constructsof the invention.

Cell-free expression systems suitable for use in the present inventionare described for instance in WO201008110, WO2010081111, WO2010083148,incorporated in their entirety herein by reference.

pylRS to be Expressed in Cell Lines According to the Invention

As used herein, pylRS relates to an amino acyl tRNA synthetase whichwill aminoacylate a suitable tRNA molecule with pyrrolysine or aderivative thereof.

The pylRS of the present invention is suitably a Pyrrolysyl-tRNASynthetase orthogonal in eukaryotic cells which is derived frommethanogenic archaea spp.—i.e. it is wildtype in methanogenic archaeaspp. or is a mutant thereof.

Preferably, the pylRS of the present invention is a Pyrrolysyl-tRNASynthetase derived from one of the following: Methanosarcina mazei (SEQID NO.1, SEQ ID NO.2, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16,),Methanosarcina barker (SEQ ID NO.3. SEQ ID NO. 17) i. Desulfitobacteriumhafniense (SEQ ID NO.4, SEQ ID NO.18). Methanosarcina acetivorans (SEQID NO.5, SEQ ID NO.19), Methanosarcina burtonii (SEQ ID NO.6, SEQ IDNO.20), Methanosarcina thermophila (SEQ ID NO.7. SEQ ID NO.21),Methanosalsum zhilinae (SEQ ID NO 8, SEQ ID NO.22), Methanohalobiumevastigatum (SEQ ID NO.9, SEQ ID NO.23), Methanohalophilus mahii (SEQ IDNO.10. SEQ ID NO.24), Desulfotomaculum gibsoniae (SEQ ID NO.11, SEQ IDNO.25), Desulfosporosinus meridei (SEQ ID NO.12, SEQ ID NO.26) andDesulfotomaculum acetoxidans (SEQ ID NO. 13, SEQ ID NO.27).

Most preferably, the pylRS of the present invention is the pyrrolysyltRNA synthetase (pylRS) derived from Methanosarcina mazei (SEQ ID NO. 1)

The pylRS of the present invention may be a wild type synthetase.

Alternatively, the pylRS of the present invention may be mutated at oneor more positions e.g. in order to increase its catalytic activityand/or to modify its selectivity for substrate amino acids (Yanagisawa2008).

Preferably, the pylRS of the present invention may be mutated atposition corresponding to Tyr 384 of SEQ ID NO.1 or its equivalent. Mostpreferably. Tyr 384 is mutated into Phenylalanine (SEQ ID NO.2).

In one embodiment, the pylRS of the present invention may be mutated atone or more positions in order to modify its substrate specificity andallow (or improve) incorporation of pyrrolysine analogs

Further mutant PylRS enzymes are described in WO09038195 and inWO2010114615 each document incorporated herein by reference in itsentirety.

tRNApyl to be Expressed in Cell Lines According to the Invention

The tRNApyl to be expressed in combination with the PylRS of the presentinvention has an anticodon and a tertiary structure which arecomplementary to the amber nonsense codon UAG, in order to function as asuppressor tRNA.

An artificial tRNA could be constructed that is complementary to othernonsense codons such as UGA, opal; UAA, ochre codons in order tofunction as a suppressor tRNA.

Thus it will be understood that although the present invention issubstantially described and exemplified by reference to use of the ambercodon for coding the nnAA and with discussion of the concept of ambersuppression, the amber codon can be replaced with an another nonsensecodon such as opal or ochre codons and would be expected to work in thesame way.

However use of amber codon is preferred.

Engineering of tRNApyl sequences in order to optimize expression ineukaryotic cell lines has been described in WO2007099854, incorporatedherein by reference.

WO2007099854 provides inter alia DNA constructs comprising a tRNA genederiving from Archaebacteria, preferably tRNApyl, a transcriptionterminator sequence placed 3′ of said tRNA gene, a promoter sequencethat induces transcription by RNA Polymerase II or III such as U1 snRNApromoter or U6 snRNA promoter placed 5′ to said tRNApyl gene.

Preferably, the tRNApyl of the present invention is a tRNApyl derivedfrom one of the following bacterial strains: Methanosarcina mazei (SEQID NO 28), Methanosarcina barkeri (SEQ ID NO 26), Desulfitobacteriumhafniense (SEQ ID NO 30), Methanosarcina acetivorans (SEQ ID NO 27).Methanosarcina burtonii ((SEQ ID NO 29), or Methanosarcina thermophila.

More preferably, the tRNApyl of the present invention is a tRNApylderived from Methanosarcina mazei (SEQ ID NO 28)

In one embodiment of the present invention, the tRNApyl is expressed ineukaryotic cells, preferably in animal cells, more preferably inmammalian cells.

In a preferred embodiment, the tRNApyl, naturally lacking promoterelements for expression in eukaryotic cells, is expressed under anexternal eukaryotic promoter.

In a particularly preferred embodiment, the external promoter is a U6promoter.

In a particularly preferred embodiment, the external promoter is an H1promoter.

In a further embodiment, the plasmid carrying the tRNApyl gene containsa transcriptor terminator sequence 3′ to the tRNApyl gene, a U6 promotersequence placed 5′ to the tRNApyl gene and a CMV enhancer region placed5′ to the promoter region.

In a particularly preferred embodiment, the insert of the plasmidcarrying the tRNApyl gene has SEQ ID NO 35.

In one embodiment of the present invention, the external promoter is arepressible promoter

In a preferred embodiment, the repressible promoter is selected from H1containing elements that allow for the repression of this promoter, suchas TetO (H1/TetO; SEQ ID NO 31), or the promoter of human U6 snRNAcontaining elements that allow for the repression of expression (e.g.U6/TetO).

It will be understood that if the stop codon indicating the end of thetarget gene is an amber codon, and it is intended to use an amber codonto encode the nnAA, then the stop codon will be changed to another stopcodon (e.g. ochre or opal). The same applies mutatis mutandis if it isintended to use another nonsense codon to encode the nnAA.

Vectors for Transformation of Eukaryotic Cell Lines with Genes EncodingpylRS and tRNApyl

The present invention provides a plasmid for efficient expression oftRNApyl in eukaryotic cells. Preferably, the tRNApyl expression plasmidincludes multiple repeats of the tRNA gene of SEQ ID 28) under a U6promoter. More preferably, the tRNApyl expression plasmid includestandem repeats of the tRNA gene of SEQ ID 28) under a U6 promoter

According to the present invention, the Pyl tRNA gene and the PylRS cDNAare carried by the same or different plasmids.

In one embodiment the tRNApyl gene and the PylRS cDNA are present on thesame plasmids.

In one embodiment the tRNApyl gene and the PylRS cDNA are present ondifferent plasmids

Vectors for Transformation of Eukaryotic Cell Lines with Genes EncodingTarget Protein

The present invention provides a vector comprising a nucleotide sequenceof the present invention optionally, operably linked to a promotersequence.

Vectors utilized in the present invention include: pJTI-Fast-DEST (Lifetechnologies), pSelect-Blasti and pSelect-Zeo vectors (invivogen), pENTRP5-P2 vector (Life Technologies), pOptivec (Life Technologies),pFUSE-CHIg-hG1 (invivogen) and pFUSE-CHLIg-hK (invivogen).

Examples of suitable promoters include, but are not limited to, CMVpromoter. SV40 Large T promoter, EF1alpha promoter, MCK promoter, andLTR promoter.

Construction of Stable Cell Lines and Selection of Stable Clones

The inventors have found that the construction of a cell line stablyexpressing the elements necessary for site specific nnAA incorporationrequires a sequential introduction of plasmids for the expression of thedifferent elements of the system (pylRS, tRNA, and target) each followedby a selection step and a sorting step (cloning step) to identify stableclones with high activity.

In one embodiment of the invention, a stable cell was obtainedexpressing all the elements for efficient nnAA introduction to serve asa starting point for the introduction of a target gene and subsequentisolation of the target protein modified at a desired position.

Suitably, this approach involves an iterative selection process wherebyan expression cassette for tRNA is first introduced into the host cellsand a pool of cells containing the constructs selected by virtue ofantibiotic resistance conferred by the vector. Next, surviving cells areselected by the introduction of a reporter construct encoding greenfluorescence protein (GFP) from Aequoria victoria containing an amberstop codon interrupting its open reading frame by transienttransfection. The selection process consists in identifying those cloneswhich, upon amber suppression, generate full length GFP whichfluorescence is quantified by flow cytometry. High functioning cellsfrom this population are thus isolated using fluorescence activated cellsorting. The best clones propagated and subsequently transfected withadditional copies of the tRNA followed by an iteration of the selectionmethod described above. The process continues with introduction of anintegrating expression construct containing cassettes for the expressionof pylRS and multiple copies of tRNA until optimal levels of expressinof each component and test amber suppressin are obtained.

Incorporation of Non-Natural Amino Acids Encoded for by Amber Codon

In proteins prepared using cell lines of the invention, one or morennAAs may be incorporated. Suitably one nnAA is incorporated into aprotein chain. In the case of the protein being an antibody, one nnAAmay be incorporated into the light chain or the heavy chain or both.

In other embodiments more than one e.g. up to four e.g. two (or perhapsthree) nnAAs may be incorporated into a protein chain. Suitably all theincorporated nnAAs are the same.

Non-natural amino acids that may be encoded by amber codon forincorporation into target proteins

The use of non-natural amino acids to allow for conjugating moieties topeptides is disclosed in WO 2007/130453, incorporated herein byreference.

As used herein an “non-natural amino acid” refers to any amino acid,modified amino acid, or amino acid analogue other than selenocysteineand the following twenty genetically encoded alpha-amino acids: alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.The generic structure of an alpha-amino acid is illustrated by FormulaI:

An non-natural amino acid is typically any structure having Formula Iwherein the R group is any substituent other than one used in the twentynatural amino acids. See, e.g., any biochemistry text such asBiochemistry by L. Stryer, 3rd ed. 1988, Freeman and Company, New York,for structures of the twenty natural amino acids. Note that thenon-natural amino acids disclosed herein may be naturally occurringcompounds other than the twenty alpha-amino acids above. Because thenon-natural amino acids disclosed herein typically differ from thenatural amino acids in side chain only, the non-natural amino acids formamide bonds with other amino acids, e.g., natural or non-natural, in thesame manner in which they are formed in naturally occurring proteins.However, the non-natural amino acids have side chain groups thatdistinguish them from the natural amino acids. For example, R in FormulaI optionally comprises an alkyl-, aryl-, aryl halide, vinyl halide,alkyl halide, acetyl, ketone, aziridine, nitrile, nitro, halide, acyl-,keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl,alkynyl, ether, thioether, epoxide, sulfone, boronic acid, boronateester, borane, phenylboronic acid, thiol, seleno-, sulfonyl-, borate,boronate, phospho, phosphono, phosphine, heterocyclic-, pyridyl,naphthyl, benzophenone, cycloalkynes such as the constrained ring suchas a cyclooctyne, cycloalkenes such as a norbornenes, transcycloalkenes,cyclopropenes, tetrazines, pyrones, thioester, enone, imine, aldehyde,ester, thioacid, hydroxylamine, amino, carboxylic acid, alpha-ketocarboxylic acid, alpha or beta unsaturated acids and amides, glyoxylamide, or organosilane group, or the like or any combination thereof.

In addition to non-natural amino acids that contain novel side chains,non-natural amino acids also optionally comprise modified backbonestructures, e.g., as illustrated by the structures of Formula II andIII:

wherein Z typically comprises OH, NH.sub.2, SH, NH.sub.2O—, NH—R′,R′NH—, R′S—, or S—R′—; X and Y, which may be the same or different,typically comprise S, N, or O, and R and R′, which are optionally thesame or different, are typically selected from the same list ofconstituents for the R group described above for the non-natural aminoacids having Formula I as well as hydrogen or (CH.sub.2).sub.x or thenatural amino acid side chains. For example, non-natural amino acidsdisclosed herein optionally comprise substitutions in the amino orcarboxyl group as illustrated by Formulas II and III. Non-natural aminoacids of this type include, but are not limited to, .alpha.-hydroxyacids, .alpha.-thioacids.alpha.-aminothiocarboxylates, or.alpha.-.alpha.-disubstituted amino acids, with side chainscorresponding e.g. to the twenty natural amino acids or to non-naturalside chains. They also include but are not limited to .beta.-amino acidsor .gamma.-amino acids, such as substituted .beta.-alanine and.gamma.-amino butyric acid. In addition, substitutions or modificationsat the .alpha.-carbon optionally include L or D isomers, such asD-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and thelike. Other structural alternatives include cyclic amino acids, such asproline analogs as well as 3-, 4-, 6-, 7-, 8-, and 9-membered ringproline analogs. Some non-natural amino acids, such as aryl halides(p-bromo-phenylalanine, p-iodophenylalanine, provide versatile palladiumcatalyzed cross-coupling reactions with ethyne or acetylene reactionsthat allow for formation of carbon-carbon, carbon-nitrogen andcarbon-oxygen bonds between aryl halides and a wide variety of couplingpartners.

For example, many non-natural amino acids are based on natural aminoacids, such as tyrosine, glutamine, phenylalanine, and the like. Thestructures of a variety of exemplary non-limiting non-natural aminoacids are provided in US 2003/0108885 A1, see the figures, e.g., FIGS.29, 30, and 31, the entire content of which document which isincorporated herein by reference.

Other examples of amino acid analogs include (but are not limited to) annon-natural analog of a Lysine or Pyrrolysine amino acid which includeone of the following functional groups; an alkyl, aryl, acyl, azido,nitrile, halo, hydrazine, hydrazide, hydroxyl, alkenyl, cycloalkenes,alkynyl, cycloalkynes, cycloalkynes such as the constrained ring such asa cyclooctyne, cycloalkenes such as a norbornenes, transcycloalkenes,cyclopropenes, aryl halide, vinyl halide, alkyl halide, aziridine,nitro, hydroxyl, ether, epoxide, vinyl ethers, silyl enol ethers, thiol,thioether, sulfonamide, sulfonyl, sulfone, seleno, ester, thioacid,boronic acid, boronate ester, borane, phosphono, phosphine,heterocyclic, pyridyl, naphthyl, benzophenone, tetrazines, pyrones,enone, imine, aldehyde, hydroxylamine, keto, thioester, ester, thioacid,organosilane group, amino, a photoactivatable cross-linker; aspin-labeled amino acid; a fluorescent amino acid; an amino acid thatcovalently or noncovalently interacts with another molecule; a metalbinding amino acid; a metal-containing amino acid; a radioactive aminoacid; a photocaged amino acid; a photoisomerizable amino acid; a biotinor biotin-analogue containing amino acid; a glycosylated or carbohydratemodified amino acid; a keto containing amino acid; an amino acidcomprising polyethylene glycol; an amino acid comprising polyether; aheavy atom substituted amino acid; a chemically cleavable orphotocleavable amino acid; an amino acid with an elongated side chain;an amino acid containing a toxic group

Non-natural amino acids suitable for use in the methods of the inventionalso include those that have a fluorescent amino acids such as thosecontaining naphthyl or dansyl or 7-aminocoumarin or 7-hydroxycoumarinside chains, photocleavable or photoisomerizable amino acids such asthose containing azobenzene or nitrobenzyl Cys, Ser or Tyr side chains,p-carboxy-methyl-L-phenylalanine, homoglutamine, 2-aminooctanoic acid,p-azidophenylalanine, p-benzoylphenylalanine, p-acetylphenylalanine,m-acetylphenylalanine, 2,4-diaminobutyric acid (DAB) and the like. Theinvention includes unprotected and acetylated forms of the above. (Seealso, for example, WO 03/031464 A2, entitled “Remodeling andGlycoconjugation of Peptides”; and, U.S. Pat. No. 6,331,418, entitled“Saccharide Compositions, Methods and Apparatus for their synthesis;”Tang and Tirrell, J. Am. Chem. Soc. (2001) 123: 11089-11090; and Tang etal., Angew. Chem. Int. Ed., (2001) 40:8, all of which are incorporatedherein by reference in their entireties).

In the present invention, non natural amino acids (nnAA) of Formula IVabove may be utilized for the production of proteins.

In an embodiment, the X group attached to the amido moeity could be analkyl azide, alkoxy azide, alkoxy epoxide, alkyl-alkyne, alkoxy alkyne,alkoxy alkene, alkyl-alkene, alkyl chain, alkyl cyclohexene, alkylcycloalkyne, alkoxyl cycloalkene, alkoxyl cycloalkyne, amidocycloalkyne, amido cycloalkene, transcycloalkene, cyclopropenes,tetrazines, pyrones, norbornenes, aryl azide, azido, a hydroxyl amine, ahydrazide, a vinyl halide, a aryl halide, a tetrazine, a pyrone, animine, boronic ester or acid, a cyano group, a carbonyl group such as analdehyde or ketone. In a preferred embodiment, non natural amino acids(nnAA) of the general structure above can have an alkyl chain from theamino acid terminus to the amido group at the opposite terminus of 1-12methylene groups.

Preferably, non natural amino acids (nnAA) of the general structureabove can contain cycloalkanes and aromatic rings as part of theconnective structure.

In an embodiment, non natural amino acids (nnAA) of the generalstructure above interact with a pyrrolysyl tRNA synthetase (PyRS) andtRNApyl. Said amino acids include:(S)-2-Amino-6-((2-azidoethoxy)carbonylamino)hexanoic acid (Lys-azide),(S)-2-Amino-6-((prop-2-ynyloxy)carbonylamino)hexanoic acid (Lys-Alkyne),S)-2-amino-6((2-oxo-2-phenylacetamide)hexanoic acid,S)-2-amino-6((2-oxo-2-propanamide)hexanoic acid,(2S)-2-amino-6-({[(2-azidocyclopentyl)oxy]carbonyl}amino)hexanoic acid,(2S)-2-amino-6-({[(2-ethynylcyclopentyl)oxy]carbonyl}amino)hexanoicacid, (2S)-2-amino-6-{[(cyclooct-2-yn-1-yloxy)carbonyl]amino}hexanoicacid,(2S)-2-amino-6-({[2-(cyclooct-2-yn-1-yloxy)ethoxy]carbonyl}amino)hexanoicacid,(2S)-2-amino-6-[({bicyclo[2.2.1]hept-5-en-2-yloxy}carbonyl)amino]hexanoicacid,(2S)-2-amino-6-[({bicyclo[2.2.1]hept-5-en-2-ylmethoxy}carbonyl)amino]hexanoicacid,(2S)-2-amino-6-{[({4-[(6-methyl-1,2,4,5-tetrazin-3-yl)amino]phenyl}methoxy)carbonyl]amino}hexanoicacid,(2S)-2-amino-6-({[(4E)-cyclooct-4-en-1-yloxy]carbonyl}amino)hexanoicacid, (2S)-2-amino-6-{[(cycloprop-2-en-1-yloxy)carbonyl]amino}hexanoicacid,(2S)-2-amino-6-{[(cycloprop-2-en-1-ylmethoxy)carbonyl]amino}hexanoicacid, (2S)-2-amino-6-{[(3-azidopropyl)carbamoyl]oxy}hexanoic acid,(2S)-2-amino-6-{[(but-3-yn-1-yloxy)carbonyl]amino}hexanoic acid,(2S)-2-amino-6-(2-azidoacetamido)hexanoic acid,(2S)-2-amino-6-(3-azidopropanamido)hexanoic acid,(2S)-2-amino-6-(5-azidopentanamido)hexanoic acid.

The nnAA will be a substrate for the pylRS.

Suitably, nnAAs of the present invention are derived from lysine.

WO2010139948 incorporated herein by reference describes several nnAAs ofinterest for the present invention, in particular the following lysinederivatives:

Other suitable nnAAs are:

Further nnAAs include:(2S)-2-amino-6-{[(3-azidopropyl)carbamoyl]oxy}hexanoic acid,(2S)-2-amino-6-1{[(3-azidopropyl)carbamoyl]oxy}hexanoic acid,(2S)-2-amino-6-{[(prop-2-yn-1-yl)carbamoyl]oxy}hexanoic acid,(2S)-2-amino-6-{[(but-3-yn-1-yl)carbamoyl]oxy}hexanoic acid,(2S)-2-amino-6-{[(prop-2-en-1-yl)carbamoyl]oxy}hexanoic acid,(2S)-2-amino-6-{[(but-3-en-1-yl)carbamoyl]oxy}hexanoic acid.

Suitably, nnAAs of the present invention are derived from(2S)-2-amino-6-hydroxyhexanoic acid.

For example:

Further non natural amino acid analogs suitable for use in the presentinvention are pyrrolysine analogs which have the structure of Formula V

whereinZ=bond, CH₂, CH—NH₂, CH—OH, NH, O, S or CH—NH₂;b is 0 or an integer 1-7; andFG=azide, alkene, alkyne, ketone, ester, aryl or cycloalkyne.In formulae V when FG represents aryl, an example is aromatic halidee.g. 4-halo phenyl such as 4-iodo phenyl.Moiety Z(CH₂)_(b)FG may, for example, represent CO-aryl e.g. CO-phenylor —COalkyl e.g. —COMe.

Exemplary compounds of formula V are the following:

Alternative pyrrolysine analogs suitable for use as non natural aminoacids in the present invention have the structure of Formula VI:

whereinZ=CH₂, CH—NH₂, CH—OH, NH, O or S;FG=azide, alkene, alkyne, ketone, ester, aryl or cycloalkyne; andb=an integer 1-4.In formulae VI when FG represents aryl, an example is aromatic halidee.g. 4-halo phenyl such as 4-iodo phenyl.

Exemplary compounds of Formula VI are:

In structures of formulae V and VI, when FG represents alkene, itsuitably represents —CH═CH₂ or —CH═CH—CH₃, preferably —CH═CH₂.

In structures of formulae V and VI, when FG represents alkyne, itsuitably represents —C≡CH or —C≡C—CH₃, preferably —C≡CH.

In structures of formulae V and VI, when FG represents ketone, itsuitably represents —C(═O)—CH₃ or —C(═O)—CH₂—CH₃, preferably —C(═O)—CH₃.

In structures of formulae V and VI, when FG represents ester, itsuitably represents —C(═O)—Oalkyl e.g. —C(═O)—Omethyl.

In structures of formulae V and VI, when FG represents aromatic halide,it suitably represents phenyl substituted by halogen, especially iodine(e.g. 4-iodo-phenyl).

In structures of formulae V and VI, when FG represents cycloalkyne, itsuitably represents cyclooctyne, e.g. cyclooct-4,5-yne.

Advantageously, the nnAAs of formulas V and VI of the present inventionhave been shown to have good incorporation as demonstrated by GFP assay.Formula VI.1 had a similar level of translational competency to FormulaV.1 in the GFP assay incorporation assay. Both the Formula V and VI areeasily modified to incorporate a variety of useful functional groupswhich can be used for site selective post translational modification.Alkynes and alkenes are readily incorporated. The pyrrolysine analogsdisclosed herein can be made using various methods. The reactionconditions can generally be determined by one of the ordinary skill inthe art.

Formula V analogs are readily prepared by the addition of an activatedcarbonyl group, such as a chloroformate, activated carboxylic acidester, isocyanate, activated carbonate or sulfonyl halide to amono-protected diamino substrate of type 1, in which the α-amino groupis protected by a protecting group (“PG”) such as a Boc, Cbz, TFA,Acetyl or Fmoc group (see Scheme 1). The coupled product 3 can undergofurther modifications, such as the displacement of halides with an azidonucleophile to install the desired functionality. Otherwise, theintermediate 3 is deprotected to remove the α-amino acid masking groupto afford the desired Formula V analog.

Formula VI analogs were prepared by conjugation of hydroxyl amino acids9 to substrates with activated carbonyls such as carboxylic acid ester,isocyanate, acid chlorides, activated carbonates or sulfonyl halides.The coupled product 11 can undergo further modifications, such as theinstallation of the azide functional group by displacement of leavinggroups such as halides or activated alcohols. The desired amino acidanalog 12 is obtained by final deprotection to remove the α-amino acidmasking group. Protecting groups may be used as per Scheme 1.

Many of the non-natural amino acids provided above are commerciallyavailable, e.g., from Sigma Aldrich (USA). Those that are notcommercially available are optionally synthesized as provided in theexamples of US 2004/138106 A1 (incorporated herein by reference) orusing standard methods known to those of skill in the art. For organicsynthesis techniques, see, e.g., Organic Chemistry by Fessendon andFessendon, (1982, Second Edition, Willard Grant Press, Boston Mass.);Advanced Organic Chemistry by March (Third Edition, 1985, Wiley andSons, New York); and Advanced Organic Chemistry by Carey and Sundberg(Third Edition, Parts A and B, 1990, Plenum Press, New York), and WO02/085923, all of which are hereby incorporated by reference.

Other nnAAs of the invention may be synthesized by published methods.For instance, synthesis of(S)-2-amino-6((prop-2-ynyloxy)carbonylamino)hexanoic acid andS)-2-amino-6((2azidoethoxy)carbonylamino)hexanoic acid is published inWO2010139948 and Nguyen et al. 2009.

S)-2-amino-6((2-oxo-2-phenylacetamide)hexanoic acid,S)-2-amino-6((2-oxo-2-propanamide)hexanoic acid,(2S)-2-amino-6-({[(2-azidocyclopentyl)oxy]carbonyl)amino)hexanoic acid,(2S)-2-amino-6-(([(2-ethynylcyclopentyl)oxy]carbonyl}amino)hexanoicacid, (2S)-2-amino-6-{[(cyclooct-2-yn-1-yloxy)carbonyl]amino}hexanoicacid,(2S)-2-amino-6-({[2-(cyclooct-2-yn-1-yloxy)ethoxy]carbonyl}amino)hexanoicacid,(2S)-2-amino-6-[({bicyclo[2.2.1]hept-5-en-2-yloxy}carbonyl)amino]hexanoicacid,(2S)-2-amino-6-[({bicyclo[2.2.1]hept-5-en-2-ylmethoxy}carbonyl)amino]hexanoicacid,(2S)-2-amino-6-{[({4-[(6-methyl-1,2,4,5-tetrazin-3-yl)amino]phenyl}methoxy)carbonyl]amino}hexanoicacid,(2S)-2-amino-6-({[(4E)-cyclooct-4-en-1-yloxy]carbonyl}amino)hexanoicacid, (2S)-2-amino-6-{([(cycloprop-2-en-1-yloxy)carbonyl]amino}hexanoicacid,(2S)-2-amino-6-{[(cycloprop-2-en-1-ylmethoxy)carbonyl]amino}hexanoicacid are disclosed in Hao, Z., Chem. Comm., 47, 4502, 2011, Schultz P G,et. al., Nat. Methods, 4, 239-244, 2007, Schultz P G, et. al., Bioorg.Med. Chem. Lett., 15, 1521-1524, 2005, Dieters A., et. al., J. Am. Chem.Soc., 125, 11782-11783, 2005, Wang, Y S, et. al., J. Am. Chem. Soc.,134, 2950-2953, 2012, Fekner, T., et. al., Angew Chem Int Ed Engl 45,1633-1635, 2009, Plass, T., et. al. Angew Chem Int Ed Engl, 51,4166-4170, 2012, Lang, K. J. Am. Chem. Soc., 134, 10317, 2012 and,Devaraj N K, Angew Chem Int Ed Engl, 48, 7013-7016, 2009.

Uses of Proteins with Incorporated Non-Natural Amino Acids

Proteins having incorporated non-natural amino acids using methodsaccording to the invention may be used for the preparation offunctionalized protein conjugates. Molecules that may be conjugated toproteins having incorporated non-natural amino acids include (i) otherproteins, e.g. antibodies especially monoclonal antibodies and (ii)polymers especially PEG groups or other groups that may cause half lifeextension in the system. Moreover these modified proteins can beconjugated to drugs or nucleotides for targeted delivery of these potentcompounds. Thus further molecules that may be conjugated to proteinshaving incorporated non-natural amino acids include (iii) cytotoxicagents and (iv) drug moieties.

More details of certain embodiments are given below in the discussion ofantibody drug conjugates.

Non-natural amino acids may conveniently contain a unique chemical grouppermitting conjugation in a targeted fashion without risk of sidereaction with other amino acids. For example non-natural amino acidsconveniently contain azide or alkyne groups permitting reaction with amolecule to be conjugated which contains a corresponding alkyne or azidegroup using the Huisgen 1,3-dipolar cycloaddition reaction.

Site Specific Conjugation

A further aspect of the invention is a process for preparing achemically modified target protein which comprises preparing a targetprotein according to the process according to an aspect of the inventionand chemically modifying the resultant target protein.

Preferred conjugation chemistries of the invention include reactionswhich are orthogonal to the natural twenty amino acids. Such reactionsdo not interact or cause side reactions with the native 20 amino acids,they are specific to the functional groups associated with the reaction.Suitably the necessary functional groups are incorporated into thetarget protein via the nnAA.

Further, said reactions proceed under conditions which are notdestructive to the protein, for instance aqueous solvents, with a pHrange which is acceptable to the protein and maintains its solubility,at a temperature which does not lead to deleterious effects upon theprotein.

Increasing the stability of the attachment moiety between the proteinand the linker can be advantageous. Conventional methods conjugate tothe thiol groups of cysteine by reaction with a maleimide forming athiol ether. The thiol ether can undergo the reverse reaction releasingthe linker drug derivative from the antibody. In an embodiment of theinvention, the conjugation chemistry employed between an azide and analkyne results in an aromatic triazole which is significantly morestable, and not as prone to reversibility.

In addition, the product of the reaction, the linkage between proteinand payload, ought to be stable, equal to or greater than the stabilityassociated with conventional linkages (amide, thiol ether). Though notan impediment to conjugation, it is often advantageous if theconjugation reactions can be done under native conditions, as this willeliminate an extra refolding processing step.

Preferred chemical conjugations for production of conjugates of theinvention include: a 3+2 alkyne-azide cycloaddition; 3+2 dipolarcycloaddition; palladium based couplings including the Heck reaction;Sonogashira reaction; Suzuki reaction; Stille coupling; Hiyama/Denmarkreaction; olefin metathesis; Diels-alder reaction; carbonyl condensationwith hydrazine, hydrazide, alkoxy amine or hydroxyl amine; strainpromoted cycloadditions, including Strain promoted azide alkynecycloaddition; metal promoted azide alkyne cycloaddition; electronpromoted cycloaddition; fragment extrusion cycloaddition; alkenecycloaddition followed by a b-elimination reaction.

According to one preferred embodiment, the incorporated amino acidcontains an azide or an alkyne group and the process of chemicalmodification comprises reacting said azide or alkyne group with areagent comprising an alkyne or azide group. The envisaged reaction is aHuisgen 1,3-dipolar cycloaddition reaction which leads to production ofa triazole linkage. The reagent comprising an alkyne or azide group maybe a protein (eg an antibody) or a toxin or a cytotoxic drug or asubstance suitable for half life extension (eg a PEG group) whichcarries an alkyne or azide group optionally via a linker.

The alkyne group of use in said reaction is, for example, a cyclooctynesuch as a bicyclo[6.1.0]non-4-yne moiety (BCN).

In a variant reaction, the incorporated amino acid contains an azide oran alkene group and the process of chemical modification comprisesreacting said azide or alkene group with a reagent comprising an alkeneor azide group. The reagent comprising an alkene or azide group may be aprotein (eg an antibody) or a toxin or a substance suitable for halflife extension (eg a PEG group) which carries an alkyne or alkene groupoptionally via a linker.

In an embodiment, conjugation chemistry of the invention is used forpreparing an antibody drug conjugate.

Chemical Modification of Product

As noted elsewhere herein, cell lines according to the invention areuseful for production of proteins containing incorporated non-naturalamino acids. Said non-natural amino acids may usefully be employed infurther chemical reactions.

A further aspect of the invention is a process for preparing achemically modified target protein which comprises preparing a targetprotein according to the process according to an aspect of the inventionand chemically modifying the resultant target protein.

Preferred conjugation chemistries of the invention include reactionswhich are orthogonal to the natural twenty amino acids. Such reactionsdo not interact or cause side reactions with the native 20 amino acids,they are specific to the functional groups associated with the reaction.

Further, said reactions proceed under conditions which are notdestructive to the protein, for instance aqueous solvents, with a pHrange which is acceptable to the protein and maintains its solubility,at a temperature which does not lead to deleterious effects upon theprotein.

According to one embodiment, the incorporated amino acid contains anazide or an alkyne group and the process of chemical modificationcomprises reacting said azide or alkyne group with a reagent comprisingan alkyne or azide group. The envisaged reaction is a Huisgen1,3-dipolar cycloaddition reaction which leads to production of atriazole linkage. The reagent comprising an alkyne or azide group may bea protein (eg an antibody) or a drug moiety (e.g. a toxin or a cytotoxicdrug) or a substance suitable for half life extension (eg a PEG group)which carries an alkyne or azide group optionally via a linker.

Optionally, the Huisgen 1,3-dipolar cycloaddition reaction can beperformed in the presence of Cu(I) catalysis.

Preferably, copper catalyzed cycloaddition reactions are carried at roomtemperature, in aqueous solution in presence of cysteine andtris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA). Alternatively,the copper catalyzed cycloaddition reactions are carried out from 4° C.to 50° C. in aqueous solution in the presence of sodium ascorbate andtris(3-hydroxypropyltriazolylmethyl)amine (THPTA). The reactions canalso be carried out in mixed aqueous/organic solution with the organiccomponent consisting of DMSO, DMF, methanol, ethanol, t-butanol,trifluoroethanol, propylene glycol, ethylene glycol and hexylene glycol.

In a variant reaction, the incorporated amino acid contains an azide oran alkene group and the process of chemical modification comprisesreacting said azide or alkene group with a reagent comprising an alkeneor azide group. The reagent comprising an alkene or azide group may be aprotein (eg an antibody) or a toxin or a substance suitable for halflife extension (eg a PEG group) which carries an alkyne or alkene groupoptionally via a linker.

When more than one nnAA is incorporated into a target protein (eg anantibody), the chemical modification may be the same or different. Forexample if two nnAAs are incorporated, one may be modified to beconjugated to a drug moiety and one may be modified to be conjugated toa PEG moiety.

Target Proteins

Target proteins include antibodies, particularly monoclonal antibodies.

Antibodies of the invention include full length antibodies and antibodyfragments including Fab, Fab2, and single chain antibody fragments(scFvs) directed to TROP-2, SSTR3, B7S1/B7x, PSMA, STEAP2, PSCA, PDGF,RaSL, C35D3, EpCam, TMCC1, VEGF/R, Connexin-30, CA125 (Muc16),Semaphorin-5B, ENPP3, EPHB2, SLC45A3 (PCANAP), ABCC4 (MOAT-1), TSPAN1,PSGRD-GPCR, GD2, EGFR (Her1), TMEFF2, CD74, CD174 (1eY), Muc-1, CD340(Her2), Muc16, GPNMB, Cripto, EphA2, 5T4, Mesothelin, TAG-72, CA9 (IX),a-v-Integrin, FAP, Tim-1, NCAM/CD56, alpha folate receptor, CD44v6,Chondroitin sulfate proteoglycan, CD20, CA55.1, SLC44A4, RON, CD40,HM1.24, CS-1, Beta2 microglobulin, CD56, CD105, CD138, Lewis Y, GRNMP,Tomoregulin, CD33, FAP, CAIX, FasL Receptor, MMPmatrix metalloproteases.

In a preferred embodiment of the invention, antibodies of the inventiondirected to tumor targets are conjugated to protein moieties selectedfrom the following: immunostimulatory and proapoptotic proteins,particularly Immune stimulators such as IL-1alpha, IL-1beta, other IL-1family members, any of the interleukins, including but not limited toIL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-13, IL-15, IL-17 family, IL-18,IL-21, IL-22, IL-23, IL-28, or costimulatory ligands such as B7.1 andB7.2, TACI. Interferons such as any of the Type I IFN family (IFN alphaand beta and lambda) or the Type II IFN gamma. Hematopoietic growthfactors such as GM-CSF. Chemokines including CXCL-1, CXCL-2, CXCL-5,CXCL-6, CXCL-8, CXCL-9, CXCL-10, and CXCL-11, CXCL-13, CCL-2, CCL-3,CCL-4, CCL-5, CCL-21, IP-10, Eotaxin, RANTES, PF4, GRO related peptides,IL-8. Proapoptotic ligands such as those of the TNF superfamilyincluding FasL, TNF, PD-L1. Antimicrobial peptides such as alpha andbeta defensins and cathelicidin LL37/hCAP18, histatins, cathepsin G,azurocidin, chymase, eosinophil derived neurotoxin, high mobility group1 nuclear proteins, HMGB1, lactoferrin. ROS and RNS producing enzymessuch as the members of NADPH oxidases (NOXs), nitric oxide synthase NOS,INOS), neutrophil granule proteins including proteases such as elastasesand cathepsins, Azurocidin (also known as CAP37 or HBP),myeloperoxidase, perforin, granzymes.

In one embodiment the target protein is an anti-Her-2 antibody.

In one embodiment, the target protein is an anti-IL-6 antibody.

In one embodiment, the target protein is an anti-PSMA antibody.

In a preferred embodiment, the anti-PSMA antibody is an scfv.

In one embodiment, the target protein is FGF21 for example having thesequence of SEQ ID No 62 or a sequence having 95% identity therewith(e.g 96, 97, 98 or 99% identity therewith). The sequence identify iscalculated taking the whole protein as the window of comparison.Conventional sequence comparing programs such as BLAST may be used.

In a preferred embodiment, FGF21 is modified to contain non naturalamino acid lys-azide or propargyl lysine at position R131 (see SEQ IDNo. 64) and conjugated to a PEG moiety via a triazole linker.

Decoy Amino Acid

A decoy amino acid of use in processes according to the invention is anamino acid derivative which is not incorporated into the extendingprotein. Alternatively, a decoy amino acid is an amino acid derivativewhich is incorporated into the extending protein but inhibits proteinelongation.

Decoy amino acids of the present invention have general Formula VII:

whereinG=H, OH, —OCH₃, OCH₂CH₃, O—C(═O)—CH₃ or NH—K-Q;X=bond, CH₂, S, O, NH, N—(C═O)— or CH-J;J=alkyl, aryl, heteroaryl or the side chain of one of the 20 naturalamino acids;Y=bond, NH, O, S, CH₂;Z=O, NH, CH₂, S, CH—NH₂;

K=CO or SO₂;

a=0, 1, 2 or 3;b=0, 1, 2 or 3;Q=—H, C₁₋₆alkyl, aryl, heteroaryl —OC₁₋₆alkyl, —OCH₂aryl,—OCH₂heteroaryl, —C₂₋₆alkenyl or —OC₂₋₆alkenyl; andR=C₁₋₆alkyl, C₂₋₆ alkenyl, —CH₂aryl, C₂₋₆alkynyl, C₁₋₆haloalkyl orC₁₋₆azidoalkyl.

An example aryl group within the definition of Q is phenyl.

Example R groups include —CH₂CH═CH₂, —CH₂CH₂Cl, —CH₂CH₂N₃, —CH₂Ph,—C(CH₃)₃, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —CH(CH₃)₂—, and —CH₂—C≡C—H.

An example aryl group within the definition of Q is phenyl.

Examples groups for Q include H, —CH₃, -Et, Ph, —OtBu, —OFmoc, —OBn,—OMe, —OEt and —OCH2CH═CH₂.

In one embodiment K is CO. In another embodiment K is SO₂.

Suitably Y represents NH. O or S and Z represents O, NH, CH₂, S orCH—NH₂ or Y represents bond, NH, O, S or CH₂ and Z represents O, NH orS.

Suitably Y represents NH, O or S. Suitably Z represents NH, O or S.Suitably Y represents NH, O or S and Z represents NH, O or S.

In one embodiment Y is NH and Z is O. In another embodiment Y is O and Zis NH.

When J represents the side chain of one of the 20 natural amino acids,examples include the side chains of cysteine, serine, threonine,aspartic acid, glutamic acid, alanine, phenylalanine, isoleucine,valine, tyrosine and tryptophan.

In an embodiment, a decoy amino acids of the invention is an amino acidsubstrate for pylRS with a chemical modified amine group, for example anN-acylated amino acid of Formula VIIA:

wherein

K is CO or SO₂;

Q=H, C₁₋₆alkyl, aryl, heteroaryl —OC₁₋₆alkyl, —OCH₂aryl,—OCH₂heteroaryl, —C₂₋₆alkenyl or —OC₂₋₆alkenyl.

Advantageously, decoy nnAAs of the present invention are able to preventthe toxic effects of amber suppression caused by the expression of thePyltRNA. The decoy prevents amber suppression by enabling thetermination of protein translation at the amber codon, in the presenceof the amber suppressor tRNA. Suitably, a decoy amino acid of FormulaVII, that lacks amino terminal group necessary to propagate polypeptidesynthesis as in Formula VIIB:

wherein

G=H;

a=4 or 5; andR=C₁₋₆alkyl, C₂₋₆ alkenyl, —CH₂aryl, C₂₋₆alkynyl, C₁₋₆haloalkyl orC₁₋₆azidoalkyl.An example aryl group within the definition of Q is phenyl.When R represents C₁₋₆azidoalkyl it suitably represents C₂₋₆azidoalkyle.g. C₂₋₄azidoalkyl.

Example R groups include —CH₂CH═CH₂, —CH₂CH₂Cl, —CH₂CH₂N₃, —CH₂Ph,—C(CH₃)₃, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —CH(CH₃)₂, and —CH₂—C≡C—H.

In one embodiment a is 4. In another embodiment a is 5.

Exemplary decoy amino acids of Formula VIIB are the following:

Based on the data in Example 12, the decoy nnAA is able to prevent thetoxic effects of amber suppression caused by the expression of thePyltRNA. The decoy prevents amber suppression by enabling thetermination of protein translation at the amber codon, in the presenceof the amber suppressor tRNA.

Decoy Protein

A decoy protein of use in process according to the invention is a benignprotein containing one or more non-natural amino acids encoded by anamber codon that is not a target.

Decoy proteins on the invention are selected from: Green fluorescenceprotein, Red Fluorescence Protein, albumin, SEAP, Actin, b-2microglobulin, glutathione-s-transferase and poly amber containingpeptide. A further example is IgG.

A decoy protein of the invention is suitably under the control of aninducible promoter selected from conditionally activated promoters andpromoter systems such as the tetracycline regulated promoters (TetO ortTA; TetOn and TetOFF), doxycycline-inducible (TRE) promoters, cAMPinducible promoters, glucocorticoid activated promoter systems, IPTGinducible promoters (lac), Cd2+ or Zn2+ inducible promoters(methalloprotein promoters), interferon dependent promoters (e.g. murineMX promoter), HIV LTR promoters (Tat), DMSO inducible promoters (globinpromoter globin LCR), hormone modulated promoters (GLVP/TAXI, ecdysone),and rapamycin inducible promoters (CID).

PEG Moieties

Target proteins may be conjugated to PEG moieties. PEG moieties may beincorporated into antibody drug conjugates. The PEG moiety may typicallyhave a molecular weight ranging between 0.5 kDa and 40 kDa e.g. 5 kDaand 40 kDa. More preferably, the PEG moiety may have a molecular weightof around 20 kDa. In addition, the PEG moieties can have a molecularweight range from 100-2000 Da. PEG moieties may be straight chain orbranched or multi armed

The PEG moieties can be functionalized with terminal alkynes, azides,cyanides, cycloalkynes, alkenes, aryl halides. The PEG can befunctionalized in such as way as to be monofunctional, homobifunctional,heterobifunctional, and multi-homofunctional.

Antibody Drug Conjugates (ADCs)

Cell lines according to the invention are particularly useful forproduction of Antibody Drug Conjugates (recombinant antibody covalentlybound by a synthetic linker to a given drug, typically a cytotoxic drug,or else a protein or a PEG group) which are homogeneous in nature, inwhich the number of drugs (or other conjugated molecule) per antibodyand position of those drugs upon the antibody are explicitly controlled,whereby monoclonal antibodies containing incorporated non-natural aminoacids are obtained and site specifically conjugated to a linker carryinga drug moiety (or other conjugated molecule) through orthogonalchemistry.

Suitably, the present invention provides a process to obtain ADCsincluding the following steps:

-   -   1. Introducing into a stable cell line of the invention one or        more plasmids carrying the DNA sequence coding for a full length        antibody, whereby a stop codon is introduced at specific        positions within the sequence    -   2. Purify the modified antibody with non natural amino acid        (nnAA) installed at desired position(s).    -   3. React a cytotoxin-linker derivative modified to include a        functional group complimentary to the nnAA installed in the        antibody with the modified antibody containing a complementary        reactive group through an orthogonal chemistry    -   4. Purify the resulting ADC

Thus, the present invention also provides ADCs whereby the antibodycomponent has been modified to incorporate non natural amino acidsbearing a unique reactive functional group at desired positions, wherebysuch functional group allows conjugation to a drug moiety (or protein orPEG group).

In an embodiment the present invention provides an antibody conjugatecomprising an anti-Her-2 antibody which is conjugated to one or moremoieties (e.g. one, two, three or four, preferably one or two,especially one) selected from protein, drug and PEG moieties via linkerscomprising a triazole moiety.

In particular, the triazole moiety may be formed by reaction of an azideor alkyne moiety in the side chain of a non-natural amino acidincorporated into the sequence of the anti-Her-2 antibody and an alkyneor azide moiety attached to the protein, drug or PEG moiety.

In one embodiment, the triazole moiety is formed by reaction of an azideor alkyne moiety in the side chain of a non-natural amino acidincorporated into the sequence of the anti-Her-2 antibody and an alkyneor azide moiety attached to the protein, drug or PEG moiety underconditions of Cu(I) catalysis.

Cu(I) catalysis is accomplished by using either a native Cu(I) sourcesuch as Copper iodide, copper bromide, copper chloride, copper thiolate,copper cyanide. The Cu(I) species can also be generated in situ by usinga copper (II) source and a reducing agent. The copper (II) source can becopper sulfate, copper (II) chloride, or copper acetate. The reducingagent can be sodium ascorbate, dithiothreitol, TCEP, b-mercaptoethanol,hydrazine, hydroxylamine, sodium bisulfite, cystamine, cysteine

Suitably, Cu(I) catalyzed cycloaddition are carried out in presence ofligands to stabilize the Cu(I) species present at the start of thereaction or generated in situ by reduction of a Cu(II) source such assodium sulfate with sodium ascorbate, including TBTA, THPTA,phenanthroline derivatives, pyridylmethanimine derivatives,diethylenetriamine, bipyridine derivatives, TMEDA,N,N-bis(2-pyridylmethyl)amine (BPMA) derivatives,N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) derivatives,trialkylamines such as triethylamine, diisopropyl ethylamine, HEPES andMES.

In another embodiment, an antibody conjugate comprises an antibody whichis conjugated to one or more moieties selected from drug and PEGmoieties via linkers comprising a triazole moiety in which the triazolemoiety is formed by reaction of an azide moiety in the side chain of anon-natural amino acid incorporated into the sequence of the antibodyand an alkyne moiety attached to the drug or PEG moiety and in which thealkyne moiety is a cyclooctyne moiety.

In another embodiment, an antibody conjugate comprises an antibody whichis conjugated to one or more moieties selected from drug and PEGmoieties via linkers comprising a triazole moiety in which the triazolemoiety is formed by reaction of an alkyne moiety in the side chain of anon-natural amino acid incorporated into the sequence of the antibodyand an azide moiety attached to the drug or PEG moiety and in which thealkyne moiety is a cyclooctyne moiety.

The cyclooctyne moiety may, for example, be a bicyclo[6.1.0]non-4-ynemoiety.

The non-natural amino acid incorporated into the sequence of theantibody is suitably a non-natural amino acid substrate for PylRS,particularly a non natural lysine analog such as(S)-2-amino-6((2-azidoethoxy)carbonylamino)hexanoic acid.

Antibodies

In the present invention ADCs include the use of full length antibodiesas well as antibody fragments such as, but not limited to Fab, Fab2, andsingle chain antibody fragments.

Antibodies suitable for conjugation to cytotoxins include those targetedagainst: anti-Her2, anti-IL-6, TROP-2, SSTR3, B7S1/B7x, PSMA, STEAP2,PSCA, PDGF, RaSL, C35D3, EpCam, TMCC1, VEGF/R, Connexin-30, CA125(Muc16), Semaphorin-5B, ENPP3, EPHB2, SLC45A3 (PCANAP), ABCC4 (MOAT-1),TSPAN1, PSGRD-GPCR, GD2, EGFR (Her1), TMEFF2, CD74, CD174 (1eY), Muc-1,CD340 (Her2), Muc16, GPNMB, Cripto, EphA2, 5T4, Mesothelin, TAG-72, CA9(IX), a-v-Integrin, FAP, Tim-1, NCAM/CD56, alpha folate receptor,CD44v6, Chondroitin sulfate proteoglycan, CD20, CA55.1, SLC44A4, RON,CD40, HM1.24, CS-1, Beta2 microglobulin, CD56, CD105, CD138, Lewis Y,GRNMP, Tomoregulin, CD33, FAP, CAIX, FasL Receptor, MMPmatrix metalloproteases.

In a preferred embodiment, antibodies of the invention are of the IgGtype.

In a particularly preferred embodiment of the invention, the antibody ismodified to comprise one or more non-natural amino acids, wherein thepositions of such non-natural amino acids are conserved amongst IgGimmunoglobulins and are selected from positions K157 of SEQ ID No 82,representing a conserved constant region of the heavy chain for an IgG,corresponding to position 274 of the anti-Her 2 antibody of SEQ ID Nos46 and 75 and position T242 of SEQ ID 82 corresponding to position 359of the anti-Her 2 antibody of SEQ ID Nos 46 and 75 and positions D70 andL81 in the framework region of the light chain of IgG, following Kabatnumbering and corresponding to D70 and L81 of SEQ ID Nos 52 and 79. Forclarity, the D70 is found in the following amino acid context:sgsrsgtdftltisslq and E81 in the following amino acid context:sslqpedfatyycqq.

One particular antibody of interest is an anti-Her-2 antibody.

The anti-Her2-antibody may, for example, have the light chain sequenceof SEQ ID No 52 or a derivative having a sequence identity of 95% (e.g.96, 97, 98 or 99%) or more thereto and having the same CDRs and theheavy chain sequence of SEQ ID No 46 or a derivative having a sequenceidentity of 95% (e.g. 96, 97, 98 or 99%) or more thereto and having thesame CDRs. The sequence identify is calculated taking the wholeantibody, but excluding the CDRs, as the window of comparison.Conventional sequence comparing programs such as BLAST may be used. ThemAb sequence described in this document has high similarity to thesequence of Herceptin. The mAb sequence utilized here was generated byplacing the antigen binding sites sequence found in Herceptin into agermline IgG1. The variable regions of the mouse antibody 4D5 directedto the extracellular domain of Her2 was generated by gene synthesisusing overlapping oligomers and cloned into a shuttle vector. Thevariable regions were then grafted onto the human frameworks encoded bypFUSE-CHIg-hG1 and pFUSE-CHLIg-hK (Invivogen) to generate a mouse-humanhybrid. Sequence comparison showed that the constructed antibody had sixamino acid substitutions relative to Herceptin. These corresponded to 5heavy chain positions and one light chain site. None of these sitescorrespond to CDR regions or sites adjacent to the CDRs.

According to an embodiment, the non-natural amino acid used forconjugation is in position 274 of the heavy chain sequence of each heavychain of said anti-Her2-antibody.

According to an embodiment, the non-natural amino acid used forconjugation is in position 70 of the light chain sequence of each heavychain of said anti-Her2-antibody.

According to an embodiment, the non-natural amino acid used forconjugation is in position 274 of the heavy chain sequence of each heavychain of said anti-Her2-antibody and also in position 70 of the lightchain sequence of each light chain of said anti-Her2-antibody.

According to an embodiment, the non-natural amino acid used forconjugation is in position 359 of the heavy chain sequence of each heavychain of said anti-Her2-antibody.

According to an embodiment, the non-natural amino acid used forconjugation is in position 81 of the light chain sequence of each heavychain of said anti-Her2-antibody.

According to an embodiment, the non-natural amino acid used forconjugation is in position 274 of the heavy chain sequence of each heavychain of said anti-Her2-antibody and also in position 81 of the lightchain sequence of each light chain of said anti-Her2-antibody.

According to an embodiment, the non-natural amino acid used forconjugation is in position 359 of the heavy chain sequence of each heavychain of said anti-Her2-antibody and also in position 70 of the lightchain sequence of each light chain of said anti-Her2-antibody.

According to an embodiment, the non-natural amino acid used forconjugation is in position 359 of the heavy chain sequence of each heavychain of said anti-Her2-antibody and also in position 81 of the lightchain sequence of each light chain of said anti-Her2-antibody.

Another particular antibody of interest is an anti-PSMA antibody,especially a scfv. The anti-PSMA antibody may, for example, have thescfv sequence of SEQ ID No 58 or a derivative having a sequence identityof 95% (e.g. 96, 97, 98 or 99%) or more thereto and having the sameCDRs. The sequence identify is calculated taking the whole antibody, butexcluding the CDRs, as the window of comparison. Conventional sequencecomparing programs such as BLAST may be used.

In a particular embodiment, an anti-PSMA scfv is modified to contain nonnatural amino acid lys-azide at position 117 (SEQ ID 60). Said scfv may,for example, be conjugated to aMMAF-valine-citruline-p-amino-benzoyl-carbonate-cycloalkyne derivative

Site Specific Modification of Antibodies for Production of ADCs

In the present invention, selection of conjugation sites for theincorporation of nnAAs into the antibody included the following steps:

Initial selection of sites was conducted using in silico predictivemethods that took into account the three dimensional structure of theantibody, its functional domains and critical amino acid residues thatplay a role in the structure or function of the antibody. Selected siteswere then screened for their physico-chemical properties and stability.

Suitably, criteria for selection of optimal conjugation sites includedthe following:

Preferred sites are: residues distal to the binding sites of theantibody; surface/solvent exposed residues (to enhance access toconjugate formation and enable efficient conjugate formation); Siteswere empirically found to allow efficient amber suppression; sites thatwere empirically found to retain the stability of the expressed proteinand conjugate

Avoided sites are: residues important for function (eg FcRN binding,FcGamma interactions), amino acid residues known to be important forfolding or structure (e.g. Cys, proline)

Six sites in human IgG1 have been identified following the criteriaoutline above. These include four heavy chain positions (T114, K274,K288 and T359) and two light chain sites (D70 and E81). HC K274, K288and T359; and LC D70, E81 were shown to efficiently incorporate nnAAsand enable conjugate formation.

Linkers

According to the present invention, the target protein or antibody maybe directly linked to the protein or drug moiety or PEG moiety or elselinked through a linker or spacer.

Linkers of the invention may be cleavable or non cleavable.

Thus the invention provides an antibody conjugate wherein the or alinker comprising a triazole moiety is a cleavable linker by virtue ofthe presence in the linker of a spacer containing a cleavage site. Thecleavage site may be an enzymatically labile cleavage site. An exampleof an enzymatically labile cleavage site is the incorporation of avaline-citrulline peptide which recognized by the enzyme cathepsin B andwhich cleaves the peptide at the citruline C-terminus

In an embodiment, the or a linker of the antibody conjugate comprises atriazole moiety that is not a cleavable linker.

The use of cleavable linkers is driven by the need for the cytotoxin tobe released within its target in an unaltered state. This is exemplifiedby cytotoxins such as monomethylauristate E <(Pettit 1997, Senter2003)>. The mechanism for release in a cleavable linker can be chemicalsuch as acid lability, or enzymatic by inclusion of a cleavable peptidewithin the linker. The mechanism can also be externally triggered by alight or other radiation source or a chemical trigger such as fluoride.

Non cleavable linkers do not have to be removed from the cytotoxin inorder to achieve the desired potency or cell killing effect duringtherapy. Thus, antibody is internalized and reduced to its amino acidcomponents in the lysosome, with the drug-linker released. It is thiscompound which requires no additional release in order to be potent.Non-cleavable linkers have no internal mechanism for releasing theintact cytotoxin, instead they rely on the benign-ness of theirinclusion on a cytotoxins framework. Non cleavable linkers can have anumber of varied structures, from relatively simple to more complexentities.

Further, linkers are defined by the manner in which they are attachedboth the cytotoxin and the antibody. For conventional approaches thisincludes chemistry for attaching to either cysteine thiols (maleimide)or lysine amines (activated acids). Linkers of the invention incorporatealkyne or azide groups.

Suitably, non cleavable linkers of the invention include a functionalhandle (Y) for attaching to the antibody at one terminus, a spacer whichbridges the two components of the ADC and provides the functional groupsnecessary to attach to the antibody and to the drug, and an thecomplimentary functional group (X) for coupling to the drug. (See FIG.43)

Suitably, the preferred functional handles are those chemical moietieswhich are complementary reactive partners to the functional group on thenon-natural amino acid installed into the target protein. The spacerportion of the molecule is a non-functional chemical bridge whichcontains the two complimentary functional groups necessary to attach tothe antibody and to the drug. In this embodiment of the linker, thisspacer has no cleavage site.

In a preferred embodiment of the invention, the functional handle (Y)includes an alkyne group. (See FIG. 44)

Preferably, the alkyne may be a terminal alkyne, an internal alkyne, acyclic alkyne and an Silyl-protected alkyne.

Preferably, the internal alkyne would contain electron withdrawinggroups adjacent to the alkyne. Preferably, the cyclic alkyne would be analkyne contained within a 7, 8 or 9 membered ring.

These electron withdrawing groups include halogens such as fluorine,bromine chlorine and iodine. Additional electron withdrawing groups ininclude hydroxyl, ethers, acetals, ketals, ketones, aldehydes,carboxylic acids, esters, nitriles, nitro, amides.

More preferably, the ring would be included in a bicyclic ring system inwhich the 8 membered ring is fused to another ring of 3, 4, 5, or 6atoms as described for instance in van Delft, F., Angew. Chem. Int. Ed,49, 1-5, 2012. M. D. Best, Biochemistry 2009, 48, 6571-6584; E. M.

-   Sletten, C. R. Bertozzi, Angew. Chem. 2009, 121, 7108-7133;-   Angew. Chem. Int. Ed. 2009, 48, 6974-6998; J. A. Prescher, C. R.    Bertozzi, Nat. Chem. Biol. 2005, 1, 13-21. J. A. Codelli, J. M.    Baskin, N. J. Agard, C. R. Bertozzi, J. Am. Chem. Soc. 2008, 130,    11486-11493. Incorporated herein by reference.

Particularly preferred cyclic alkynes are described in U.S. Pat. No.7,807,619 and U.S. Ser. No. 12/049,034, incorporated herein byreference.

Particularly preferred bicyclic alkynes are bicyclononynes as describedin WO2011/136645 (incorporated herein by reference):

In an embodiment, a non cleavable linker of the invention may contain analkene as functional handle (Y) at the antibody attachment site.

Suitably, the alkene can be mono, di, tri or tetra substituted.

Suitably, the alkene can be incorporated as part of a ring.

In the preferred embodiment, the alkene can be part of a 3-12 memberedring.

In a further preferred embodiment, the alkene can be part of a bicyclicring system such as norbornene, bicyclic furan or bicyclic pyrrolesystem. (See FIG. 45)

Preferably, the functional handle (Y) at the antibody attachment siteincludes a vinyl halide.

Preferably, the vinyl halide includes a halide such as fluorine,chloride, bromine, or iodine at either the Z or Y positions or both.Furthermore, the vinyl halide can be terminal in which the R-group is ahydrogen. The vinyl halide group can also contain additionalsubstitution at the R position, including alkyl and aryl groups,carbonyl groups.

Preferably, the vinyl halide is part of a cyclic compound.

More preferably, the vinyl halide is part of rings with 3, 4 and 5atoms.

In an embodiment, the functional handle (Y) at the antibody attachmentsite includes a reactive aromatic ring substituted with a silyl groupand either a halide or triflate, tosylate or mesylate at the LGposition. (See FIG. 46)

In a further embodiment, the functional handle (Y) at the antibodyattachment site includes a reactive azide group at the terminus. (SeeFIG. 47)

Suitably, a cleavable linker of the invention includes a functionalhandle (Y) for attaching to the antibody at one terminus, a spacer andan the complimentary functional group (X) for coupling to the drug. (SeeFIG. 48)

Suitably, cleavable linkers of the ADCs of the invention include acleavage site.

Suitably, the cleavage site may be triggered enzymatically, chemically,or externally.

In an embodiment, the cleavage site is placed at the drug attachmentsite.

In an alternative embodiment, the cleavage site is at the drugattachment site. (See FIG. 49)

In an embodiment, the cleavable linker includes a functional handle (Y)at the antibody attachment site with an alkyne.

Preferably, the alkyne may be a terminal alkyne, an internal alkyne, acyclic alkyne and an Silyl-protected alkyne.

Preferably, the internal alkyne would contain electron withdrawinggroups adjacent to the alkyne. These electron withdrawing groups includehalogens such as fluorine, bromine chlorine and iodine. Additionalelectron withdrawing groups in include hydroxyl, ethers, acetals,ketals, ketones, aldehydes, carboxylic acids, esters, nitriles, nitro,amides.

Preferably, the cyclic alkyne would be an alkyne contained within a 7, 8or 9 membered ring.

More preferably, the ring would be included in a bicyclic ring system inwhich the 8 membered ring is fused to another ring of 3, 4, 5, or 6atoms.

In an alternative embodiment, the cleavage site and spacer are reversedin order with the cleavage site is at the drug attachment site. (SeeFIG. 50)

In an embodiment, a cleavable linker of the invention may contain analkene as functional handle (Y) at the antibody attachment site.

Suitably, the alkene can be mono, di, tri or tetra substituted.

Suitably, the alkene can be incorporated as part of a ring.

In a preferred embodiment, the alkene can be part of a 3-12 memberedring.

In a further preferred embodiment, the alkene can be part of a bicyclicring system such as norbornene, bicyclic furan or bicyclic pyrrolesystem.

Preferably, the functional handle (Y) at the antibody attachment siteincludes a vinyl halide. (See FIG. 51)

Preferably, the vinyl halide includes a halide such as fluorine,chloride, bromine, or iodine at either the Z or Y positions or both.Furthermore, the vinyl halide can be terminal in which the R-group is ahydrogen. The vinyl halide group can also contain additionalsubstitution at the R position, including alkyl and aryl groups,carbonyl groups.

Preferably, the vinyl halide is part of a cyclic compound.

More preferably, the vinyl halide is part of rings with 3, 4 and 5atoms.

In an alternative embodiment, the cleavage site and spacer are reversedin order with the cleavage site is at the drug attachment site

In an embodiment, the functional handle (Y) at the antibody attachmentsite includes a reactive aromatic ring substituted with a silyl groupand either a halide or triflate, tosylate or mesylate at the LGposition. (See FIG. 52)

In an alternative embodiment, the cleavage site and spacer are reversedin order with the cleavage site is at the drug attachment site.

In a further embodiment, the functional handle (Y) at the antibodyattachment site includes a reactive azide group at the terminus. (SeeFIG. 53)

In an alternative embodiment, the cleavage site and spacer are reversedin order with the cleavage site is at the drug attachment site

In an embodiment of the present invention, the spacer portion of bothcleavable and non-cleavable linker can be structurally diverse andinclude alkyl chains, alkyl rings, aromatic rings, aniline derivativeincluding p-amino-benzyl carbonate, alkenes, polymers such aspolyethylene glycol.

In a preferred embodiment of the invention, the linker is composed of acycloalkyne at one terminus for attachment to the antibody viaazide-alkyne cycloaddition. Attached to the cycloalkyne is carbon chainwhich is then attached to a valine-citrulline peptide. The C-terminus ofcitrulline is coupled to a p-amino-benzoyl carbamate (PABC). This isturn is connected to the N-terminus of MMAF. (See FIG. 54) Thisvaline-citrulline peptide is recognized by the enzyme cathepsin B, whichcleaves the peptide at the citrulline c-terminus. Follow the cleavage,the p-aminobenzoyl undergoes an elimination reaction to extrude CO2 andthe MMAF group. Thus, the entire cyclo-alkyne-val-cit-PABC combinationis a cleavable linker.

In an embodiment of the invention, the linker releases the drug from theADC upon a trigger

Suitably, a trigger may be found near or within the target cell.

Preferably, the linker is found within the cell. Suitably anintracellular trigger includes an enzymatic trigger.

Suitably, enzymatic cleavage sites include amino acid sequencesspecifically recognized by intracellular enzymes.

Preferred enzymatic cleavage site of the invention are Cathepsin(Valine-Citrulline) and Furin (Arg-N-Arg-Arg),

Alternatively, chemical triggers are found within the target cell.

Suitably, chemical triggers include acid hydrolysis of chemical moietiesincluding, esters, amides, acetals, ketals, nitriles, ether cleavage,carbamates, ureas, sulfonamides, sulfonyl, sulfenyl, phosphinamides,phosphoramidates, enamines, imines, silyl ethers, ortho esters,boronates.

Alternatively, chemical triggers can also include reduction of chemicalmoieties including disulfides, fluoride addition to silyl groups,reverse cycloadditions and reverse Michael additions.

Alternatively, release of the drug from the linker can be achieved byextracellular stimuli such as exposure to radiation of a particularwavelength.

Drug Moieties

Drug moieties of the present invention, such as cytotoxin drug moieties,include small molecules, natural products, synthetically derived drugs,proteins such as immunotoxins, and radionuclides.

In an embodiment, the drug moiety is an auristatin moiety eg auristatinor a derivative thereof such as monomethyl auristatin E (MMAE)(Vedotin)or monomethyl auristatin F (MMAF), Auristatin F (AF), Amanitin,Paclitaxel and doxorubicin.

Other drug moieties include maytansine, paclitaxel, doxorubicin andimmunotoxins such as exotoxin or bouganin as well as radionuclides suchas Iodine-131, Yttrium-90, Samarium-135, and Strontium-89 which may alsobe incorporated into organic molecules. (see for instance: MMAE: Senter,P E, et. al, BLOOD, 102, 1458-1465. MMAF: Senter, P E, et. al., Bioconj.Chem. 2006, 17, 114-124. Maytansine: Lewis-Phillips G D, Cancer Res.,63, 9280-9290, 2008. Bouganin: MacDonald G C, et. al, J. Immunotherapy,32 574-84, 2009.

Most suitably the drug moiety is a moiety selected from a doxorubicin,paclitaxel and auristatin moiety.

Salts

Amino acids, amino acid derivatives, decoy amino acids and pyrrolysineanalogs described herein may optionally be employed in the form of asalt. Any such salts form an aspect of the invention. Salts ofcarboxylic acids may include salts formed with Group 1 and Group 2metals, especially soluble salts such as sodium and potassium salts.Salts of amines may include salts formed with weak and strong acids,such as HCl, HBr or acetic acid.

EXAMPLES Example 1: Generation of a Stable Cell Line

The generation of a platform cell line capable of site specificintegration of nnAAs into a target protein required the stepwiseconstruction of a cell line stably expressing the pylRS and the pyltRNA.This was accomplished by sequential introduction of the pylRS/tRNAexpression elements and iterative selection steps to identify highfunctioning cells (FIG. 1).

A plasmid containing nine copies of the U6-pyltRNA expression cassetteas well as a sequence encoding the human INF□ Matrix Attachment Region(pSB-9xtRNA-MARS whereby the U6 is defined in SEQ ID 32, the tRNAsequence in Seq ID28), a sequence element that mediates the organizationof chromatin in the nucleus and plays a role in the regulation of geneexpression and enhances stability of these elements through replication(Klar 2005; Heng 2004; Piechaczek 1999) were transfected into DG44-CHOcells and selected in ProCHO4 medium supplemented with HT supplementcontaining 0.1 mM hypoxanthine and 0.016 mM thymidine, 8 mM glutamine, 5ug/mL blasticidin (ProCHO4-C). Cells were then selected for tRNAfunction using a GFP reporter assay and cell sorting. Briefly, cellswere transfected with pJTI-R4 PylRS eGFPY40, encoding a FLAG taggedpylRS (SEQ ID 2) and the reporter eGFP containing an amber codon inplace of the codon encoding tyrosine at position 40 (eGFPY40, SEQ ID 38,and cells exposed to 2 mM nnAA ALOC (Nε-Allyloxycarbonyl-L-Lysine) for14 h. 30,000 cells showing the highest levels of fluorescence werecollected into fresh ProCHO4-C medium using a BD FACS Aria II cellsorter and expanded. This population of cells represents a sorted poolcontaining pyltRNA activity. To test whether the sorted pool showedimproved function over the parental, pre-sorted pool, both populationswere transiently transfected with pJTI-R4 PylRS eGFPY40 a GFP control(pTracer EF/HisA; Life technologies modified to contain F64L and S65Tmutations) encoding a wild type eGFP (SEQ ID37). Transfected cells weregrown for 24h in ProCHO4-C medium containing 2 mM ALOC and analysedusing a Accuri flow cytometer and the fluorescence levels quantified inthese and control cells (FIG. 2A) These data show that the sorted cellpopulation has higher amber suppression efficacy in the presence of nnAAas compared to the parental strain or untransfected controls. Thisintermediate cell population is referred to as DG44-CHO-191. While theDG44-CHO-191 cells were capable of amber suppression their efficacy waslimited, with less than 43% amber suppression based on the GFPY40. Thelevels of tRNA were shown to be the limiting factor in the efficacy ofamber suppression. Therefore, sorted DG44-CHO-191 cells were transfectedwith pSZ-9xtRNA and cells selected in DMEM-BZ (DMEM (Life Technologies),2 mM glutamax, 1 mM sodium pyruvate, 6 mM glutamine, 1× non essentialamino acids (Gibco CAT#11140-050), 10% fetal bovine serum, HTsupplement, 5 ug/ml Blasticidin, 0.5 mg/mL Zeocin). The surviving cellpool, referred to as DG44-CHO-200-12, and DG44-CHO-191 cells weretransfected with pJTI-R4 PylRS eGFPY40 or pTracer. Cells containingadditional copies of the tRNA expression cassettes demonstrate increasedeGFPY40 dependent fluorescence and thus amber suppression efficacy (FIG.2B). These data show that the stepwise, iterative selection and cellsorting methodology results in the identification of cells with improvedfunction. With the understanding that tRNA is a limiting component ofthe system, and to further increase the expression levels of the pyltRNAand thus efficacy of amber suppression of this cell population,DG44-CHO-200-12 cells were subjected to cell sorting to isolate cellswith high amber suppression capabilities. Here DG44-CHO-200-12 cells(Containing pSB-9x-MARS and pSZ-9x) were transiently transfected withpJTI-R4-pylRS-eGFPY40 and cells grown in medium containing 2 mM ALOC.7,000 cells showing the highest 1% fluorescence levels were isolatedusing the BD FACS Aria II cell sorter and propagated in DMEM-BZ. Thisresulted in the cell pool referred to as DG44-CHO-208-2.

The completion of the platform cell line required the stableintroduction of a cassette for the expression of pyrlRS. Thus, 208-2cells were transfected with pMOAV2 or pMOAV2-puro carrying the cDNAsequence coding for pylRS of SEQ ID No 2 (Y384F mutant) or SEQ ID 1(WT), and transformants selected in DMEM-BSD-Zeo containing 0.5 mg/mLhygromycin (DMEM-HBZ), or DMEM-BZ containing 7.5 ug/ml puromycin(DMEM-PBZ), to generate a selected pool of cells called DG44-CHO-211-1(hygro) or DG44-CHO-211-2 (puro). Antibiotic resistant cells werecultured and transfected with pENTR-P5-P2 eGFPY40 encoding the eGFPreporter construct and cells cultured in the presence of 2 mM ALOC for 1hour and 20 min and subsequently cells showing high fluorescence levelswere isolated using cell sorting. Here, 1331 cells (from 1,712,332events) of the 211-1 and 1169 of 211-2 were isolated. The sortedpopulations called DG44-CHO-223-1 or DG44-CHO-223-2 were cultured inDMEM-HBZ or DMEM-PBZ.

To determine whether sorting of pylRS containing populations improvedthe efficacy of amber suppression transient transfections ofDG44-CHO-223-1 and its parental cell line DG44-CHO-211-1 were conductedwith a reporter plasmid encoding GFP containing an amber codoninterrupting its open reading frame (P2-P5 eGFPY40), eTracer, or leftuntransfected. Transfected cells were incubated with 2 mM ALOC for 28 hand fluorescence quantified by flow cytometry utilizing an Accuri flowcytometer (FIG. 2C). While DG44-CHO-211-1 and DG44-CHO-223-1 showedequivalent transfectability (eTracer control), DG44-CHO-223-1 showed agreater than 5-fold more eGFPY40 dependent fluorescence than theparental cell line. This result indicates that sorting cells enables theisolation of highly active amber suppressing cells and the isolation ofan efficient platform cell line.

Next, the platform cell line was used to develop an expression cell linecontaining a stably integrated gene target coding for the protein to bemodified with a nnAA.

The DG44-CHO-223-2 cell line was transfected with pOtivec-28D2amb274plasmid containing genes for the expression of an IgG directed againsthuman IL-6 with an amber codon at position K274 of the heavy chain cDNA.To do this an antibody directed against the human cytokine IL-6 wasgenerated by grafting the Variable regions of a rabbit antibody directedagainst the human cytokine IL-6 were grafted onto a human frameworks byPCR amplification and cloning into the vectors pFUSE-CHIg-hG1 (heavychain, SEQ ID 40) and pFUSE-CHLIg-hK (Light chain, SEQ ID 44)(Invivogen)to generate a rabbit-human hybrid, as described in WO2012032181,incorporated in its entirety herein by reference. An amber codon wasintroduced at position K274 of the heavy chain constant region by sitedirected mutagenesis (SEQ ID 42). Clones containing the amber codonswere identified by DNA sequencing. To generate an integrating constructthis IgG, the promoters and ORF for the heavy chain was amplified by PCRand cloned by restriction enzyme digestion and ligation into pOptivec(Life technologies). The light chain and a single copy of the tRNA werejoined by two step PCR method using overlapping oligomers and clonedinto available sites into the pOptivec plasmid containing the heavychain. The resulting vector was introduced by transfection into theplatform cell line DG44-CHO-223-2 and cells selected by growth of theculture in growth medium lacking hypoxanthine and thymidine, DMEM-HT(DMEM, 2 mM glutamax, 1 mM sodium pyruvate, 6 mM glutamine, 1× nonessential amino acids (Gibco CAT#11140-050), 10% dialyzed fetal bovineserum, 5 ug/ml Blasticidin, 0.5 mg/mL Zeocin, 0.75 ug/mL Puromycin). TheOptivec vector also contains the gene for dihydrofolate reductase(DHFR), which enables growth of DG44 CHO cells in medium lacking HTsupplements and in the presence of methotrexate. Cells were furtherselected in medium lacking DMEM-HT and containing 10 nM, 50 nM, and 100nM methotrexate (MTX). Live cells were harvested and distributed at 50cells/well into 96 well trays in the same medium with half theantibiotic concentrations used previously outlined. In the absence of annAA in the growth medium, the pylRS/tRNA pair is inactive and ambersuppression does not occur. Thus, a truncated IgG heavy chain isexpressed and secreted into the growth medium. After 10-12 days, wellswere monitored for growth and ELISA assays used to identify wells whichcontain colonies that express high levels of truncated IgG. To do thisELISA plates were coated with 1 ug/mL 3×FLAG-IL-6-Avi in phosphatebuffered saline (PBS) for 1 h or overnight at 4 C. After washing inwater and blocking in PBS containing 1% BSA, 15 ul of expression mediumwas diluted with 35 ul of PBS containing 0.1% skim milk and added toeach of the wells for 1 h at room temperature. Wells were washed inwater several times and 50 ul of a 1:10,000 dilution of the secondaryantibody conjugated to horse raddish peroxidase (anti-human H+L-HRP;Jackson Laboratories) for 1 h at room temperature. Wells were thenwashed and 50 ul of Sureblue Reserve TMB (KPL) added to each well. After5-10 minutes 0.1N H2SO4 was added to stop the reaction and colordevelopment quantified using a plate reader at 450 nM wavelength. Wellscontaining cells that expressed high levels of the truncated IgG werepropagated and expanded. This assay led to the identification of sevenclones showing high truncated IgG expression. To determine if theisolated clones showed efficient amber suppression, the clones wereexposed to 2 mM ALOC and the expression levels of the full length IgGwere measured by ELISA. Briefly, a goat human anti-FC antibody (Jacksonlabs) was used to specifically capture full length IgG, and nottruncated IgG. Out of the seven clones tested, one showed high levels offull length expression (3F2, SEQ ID 42). To demonstrate the efficiencyof amber suppression, the 3F2 clone was utilized for the expression andpurification of IgG. The 3F2 clone was cultured to 90% confluence in atissue culture flask in medium containing 50 nM MTX and cells incubatedwith 2 mM lys-azide (nnAA) in expression medium (DMEM, 2 mM glutamax, 1mM sodium pyruvate, 6 mM glutamine, 1× non essential amino acids (GibcoCAT#11140-050), 10% low IgG fetal calf serum, 5 ug/ml Blasticidin, 0.5mg/mL Zeocin, 0.75 ug/mL Puromycin). Cells were allowed to expressantibody for 7 days and medium harvested. Antibody from the expressionsupernatant was captured on a protein A column and washed with PBS.Bound protein was eluted in 50 mM glycine pH3.0 and peak fractionscontaining the IgG dialyzed to PBS. Purified antibody containing alys-azide nnAA are referred to as AzAb. Representative samples wereresuspended in SDS-PAGE loading buffer and 0.5 ug and 1 ug respectivelyresolved by SDS-PAGE under reducing and non reducing conditions andstained with coomassie blue (FIG. 3A). To demonstrate that the expressedproduct contained a nnAA (lys-azide) the expressed protein was incubatedwith a 100 fold excess of 20 KDa-PEG containing a cyclic alkynefunctional group, for 4 h at room temperature. Equal amounts of thestarting material and the PEG-IgG conjugate were resolved by SDS-PAGEand visualized by coomassie staining (FIG. 3B). The PEG alters themolecular weight of the conjugate resulting in a retardation of gelmobility. When the reaction mixture was resolved under denaturing andreducing conditions, it was observed that only the heavy chain of theIgG, which was designed to contain the nnAA integration site (atposition 274) shows a gel mobility shift. In contrast, the light chaindoes not appear to be altered by the conjugation reaction. These datademonstrate that the expressed protein contains a moiety that isspecifically modified and that the conjugation conditions are specificto the heavy chain. To further demonstrate that the mobility shiftobserved with the conjugate represents PEGylation of the IgGanti-IL-6AzAb, a control IgG, the starting material for the conjugation reactionand the conjugated IgG were bound to protein A. The bound material waswashed with PBS to remove unconjugated PEG, and protein eluted with 2%SDS. This material was then resolved by SDS-PAGE under reducingconditions and proteins visualized by Coomassie-blue staining, iodinestaining to visualize PEG, and Western blotted using an anti-human FCspecific antibody (Jackson labs) to detect the heavy chain (FIG. 3C).These data show that the conjugate is formed specifically at the heavychain and that the molecular weight increase is due to the formation ofthe conjugate with PEG.

A second clone expressing an antibody to IL-6 was identified andcharacterized in parallel (7B1, SEQ ID 42) as was a clone generated asindicated above for an antibody directed against her2/neu (3E9, SEQ ID48), containing an amber codon encoded into the heavy chain at the sameposition described above (K274, SEQ ID 48). The expression levels ofthese cell lines was quantified and a per cell production determined inexpression medium in the presence of lys-azide (FIG. 3D). These datademonstrate the applicability of the present process to the expressionof different antibodies containing nnAAs.

Example 2: Amber Suppression Associated Toxicity

During the course of the platform cell line isolation the inventorsobserved that as an increasing amount of the pylRS/tRNApyl wasintroduced in order to improve the efficiency of the system, theviability of the cells deteriorated. In particular increasing tRNApyllevels were found to have the greatest impact on amber suppressionefficacy. This was observed in cells transiently transfected withpJTI-R4-pylRS-eGFPY40 and vectors encoding different numbers of U6-tRNAexpression cassettes and the mean fluorescence was determined using anAccuri flow cytometer (FIG. 4A). We observed that cells lacking a tRNAexpression cassette, or cells grown in the absence of ALOC did not showa significant GFP signal. However, the expression level of GFP increasedwith the number of tRNA gene copies indicating that tRNA is an importantcomponent of the amber suppression system. To further refine the effectof tRNA levels in amber suppression we transiently transfected differentamounts of vectors encoding pylRS or tRNA genes and gauged the efficacyof amber suppression on a target protein containing an amber stop codonin the presence of 2 mM ALOC. When an expression construct encoding thehuman cytokine, FGF21 containing an amber codon at amino acid residue131 (where the initiator methionine is 1—SEQ ID63, SEQ ID 64), was cotransfected with pylRS and 6 gene copies of the U6-tRNA cassette weobserved an approximately 50% conversion of truncate to full lengthFGF21 (FIG. 4B). Doubling the amount of pylRS vector in the transfectiondid not significantly alter the ratio between truncated and full lengthFGF21 (2×pylRS). However, introducing additional copies of the tRNAcassettes (15×U6-tRNA) resulted in a significant increase in therelative expression of full length FGF21. This indicated that the tRNAlevels were the greatest limiting factor to amber suppression.

Thus, the generation of a cell line with efficient amber suppressionproperties requires high levels of tRNA expression. However, theinventors found that the expression of the tRNA plasmids in particularwere deleterious to cell growth. The inherent toxicity of the tRNAexpression cassettes was reflected in observable morphological changesand decreased growth rates of high functioning platform cells ascompared to the parental lines.

While the efficacy of the platform cell lines is impacted directly bytRNA levels, high levels of tRNApyl led to cytotoxic effects. Theinventors observed that while introduction of high numbers of U6-tRNAgenes improved amber suppression in the presence of a nnAA and thepylRS, high levels of the tRNA also led to cytotoxicity. To confirmwhether this effect was associated with tRNA expression a CHO cell lineselected for the presence of pSB-9xtRNA (DG44-CHO-191) was transientlytransfected with a vector encoding eGFPY40 (P5-P2 eGFPY40) alone or incombination with a vector encoding pylRS under control of the CMVpromoter (pCEP4-pylRS), or a vector containing nine copies of theU6-tRNA in a plasmid also containing a pOriP element (pOriP-9xtRNA) andallows for prolonged retention of the plasmid in cells expressing EBNA-1(Shan 2006 and EP1992698 incorporated herein in its entirety byreference), and the cells incubated at 37 C for 48h. The fluorescencelevels were quantified using a flow cytometer (FIG. 4C). While theaddition of pylRS expression cassettes in a background of cellsexpressing pyltRNA leads to an increased level of amber suppression inthe absence of a nnAA, this effect was amplified in cells transfectedwith additional copies of the tRNA. These data suggest that high levelsof the tRNApyl induce background amber suppression levels well abovewild type cells. The toxicity associated with amber suppression isdocumented in the literature for various systems and is largelyattributed to the extension of essential genes that normally terminatein amber codons (Liebman and Sherman 1976; Liebman et al., 1976). It iscurrent thinking that the extension of these genes beyond their naturalstop, can alter, decrease or eliminate the function of these proteins.Finally, to determine whether amber suppression led to cytostaticeffects, 1000 HEK293 c18 cells plates in a 96 well plate and transientlytransfected with pCEP4-pylRS and pOriP-9xtRNA constructs. Cells weregrown in DMEM-C medium containing a titration of nnAA (ALOC) startingwith 5 mM to 0.08 mM. Cell viability was assayed at the time of thetransfection, and after 5 days growth using an MTS colorimetric assays(FIG. 4D). The data show that even small concentrations of the nnAA ledto a cytotstatic effect. Upon the current hypothesis, the toxicityassociated with amber suppression is inherent to the system and cannotbe avoided. Thus, a platform cell line containing tRNApyl and PrylRS isnot suitable for manufacturing of protein based drugs which require highproductivity as measured by amount of protein produced per cell.

However, upon transfection of a target protein containing an ambercodon, and subsequent selection, the inventors observed that the cellsregained a spindle shape and flattened appearance that is characteristicto untransfected cells and showed an improved growth rate (FIG. 4E).

This suggests that the presence of high levels of a message containingan amber codon absorbs the background amber suppression and limits theimpact upon essential genes. Thus, the construction of a cell line mayrequire a preexisting and high expressing target containing an ambercodon that would enable the isolation of very high functioning ambersuppressing cells.

Example 3—Techniques to Mitigate Amber Suppression Associated Toxicity

The toxicity associated with amber suppression led us to conceive ofalternative approaches that would mitigate this toxicity in thedevelopment of an expression cell line while enabling the isolation ofhighly active amber suppressor cells.

“Target First” Approach

One approach to mitigate the observed toxicity in the development of astable expression cell line while enabling the isolation of highlyactive amber suppressor cells requires the introduction of a highlyexpressed target gene that contains an amber codon prior to theintroduction of the pylRS and pyltRNA. High levels of message from thisgene effectively compete with endogeneous gene expression for theactivated pyltRNA available in the cell and thus reducing the impact tothe cell's functional machinery. To do this an eukaryotic expressionhost cell is transfected with a gene intended for expression andcontaining one or more amber stop codons, such as an IgG cloned into thevector pOptivec (Life Technologies). Transfected cells are selected byvirtue of their resistance to, and ability to grow in medium lacking HTand in medium lacking HT and supplemented with 10 nM, 50 nM or 100 nMMTX. Surviving cells are cloned by transferring 1-50 cells to each wellof a 96-well plate and allowed to populate the well. Wells are then beassayed by ELISA to identify wells containing high titers of truncatedantibody. For this, ELISA plates are coated with antigen (for example 1ug 3×FLAG-IL-6-Avor 0.5 ug/mL Her2 extracellular domain) in phosphatebuffered saline (PBS) for 1 h or overnight at 4 C. After washing andblocking in PBS containing 10% goat serum or 1% BSA, 40 ul of PBScontaining 10% goat serum or 35 ul of 1% skim milk and 10 ul ofexpression medium are added to appropriate wells for 1 h at roomtemperature. Wells are washed in water several times and 50 ul of a1:4,000 dilution of the secondary antibody conjugated to horse radishperoxidase (anti-human Kappa-HRP) (Jackson Labs) are added to each wellfor 1 h at room temperature. Wells will then be washed and 50 ul ofSureblue Reserve TMB (KPL) added to each well. After 5-10 minutes colordevelopment is stopped by the addition of 0.1N H2SO4 and colorgeneration quantified using a plate reader at 450 nM wavelength. Acontrol IgG of known concentration is used to establish a standardcurve. Wells containing cells that expressed high levels of thetruncated IgG are propagated and expanded.

Functional elements for the introduction of nnAAs are introduced andselected either sequentially or concurrently. In one example, cellsshowing high expression of the target gene are transfected with pMOAV2or pMOAV2puro, containing genes for pylRS and pyltRNA. Transfected cellsare selected in DMEM containing 2 mM glutamax, 1 mM sodium pyruvate, 6mM glutamine, 1× non essential amino acids (Gibco CAT#11140-050), 10%fetal bovine serum, and 0.5 mg/mL hygromycin or 7.5 ug/ml puromycin.Surviving cells are propagated and 1-50 cells from this populationseeded into each well of 96 well plates. Once the cells have expandedand colonies form, cells are exposed to nnAA at 2 mM and functionallyassayed using ELISA assays to identify clones with amber suppressionefficiencies greater than 40% or 50% or preferably greater than 60 or80% or 90%. To quantify full length IgG expression ELISA plates arecoated with 1 ug/mL anti-human FC (Jackson Labs) antibodies in phosphatebuffered saline (PBS) for 1 h or overnight at 4 C. After washing andblocking in PBS containing 10% goat serum, 40 ul of PBS containing 10%goat serum and 10 ul of expression medium are added to appropriate wellsfor 1 h. at room temperature. Wells are washed in water several timesand 50 ul of a 1:10,000 dilution of the secondary antibody conjugated tohorse raddish peroxidase (anti-human H+L-HRP)(Jackson Labs) is added toeach well for 1 h at room temperature. Wells will then be washed and 50ul of Sureblue Reserve TMB (KPL) added to each well. After 5-10 minutescolor development is stopped by the addition of 0.1N H2SO4 and colorgeneration quantified using a plate reader at 450 nM wavelength. Acontrol IgG of known concentration is used to establish a standardcurve. This assay will determine the expression levels of full lengthIgG. To determine truncated IgG levels, ELISA plates are coated withantigen, for example 1 ug/mL 3×FLAG-IL-6-Avor 0.5 ug/mL Her2extracellular domain in phosphate buffered saline (PBS) for 1 h orovernight at 4 C. After washing and blocking in PBS containing 1% BSA,35 ul of PBS containing 0.1% skim milk and 15 ul of expression mediumare added to appropriate wells for 1 h. at room temperature. Wells arewashed in water several times and 50 ul of a 1:10,000 dilution of thesecondary antibody conjugated to horse raddish peroxidase (anti-humankappa-HRP)(Jackson Labs) are added to each well for 1 h at roomtemperature. Wells will then washed and 50 ul of Sureblue Reserve TMB(KPL) added to each well. After 5-10 minutes color development isstopped by the addition of 0.1N H2SO4 and color generation quantifiedusing a plate reader at 450 nM wavelength. A control IgG of knownconcentration is used to establish a standard curve. The ratio oftruncated to full length IgG in each well is determined and wellsshowing high amber suppression activity, where the full length IgGlevels are at least 25 or 50%, preferably 40-60% or 80-90% or greater ofthe total produced IgG are propagated.

If necessary, additional tRNA genes will be introduced into theseselected pools of cells to further improve the efficacy of ambersuppression. To do this, pSB-9xtRNA-MARS expression cassette istransfected into these cells and transfectants selected by virtue ofantibiotic resistance in DMEM containing 2 mM glutamax, 1 mM sodiumpyruvate, 6 mM glutamine, lx non essential amino acids (GibcoCAT#11140-050), 10% fetal bovine serum, and 0.5 mg/mL hygromycin or 7.5ug/ml puromycin containing 5 ug/mL blasticidin (DMEM-BSD) oralternatively ProCHO4 (Lonza) or equivalent medium containing, 8 mMglutamine, 0.5 mg/mL hygromycin or 7.5 ug/ml puromycin and 5 ug/mLblasticidin (ProCHO4-BSD). Cells with the highest activity of the tRNAare selected using the ELISA assays described above to determine fulllength and truncated IgG production yields in cells exposed to nnAA.Cells showing improved full length IgG to truncated IgG ratios overparental cells are propagated. If additional tRNA gene insertions arerequired the process is repeated as described above with pSZ-9xtRNA andcells selected in medium containing 5 ug/mL Zeocin followed by afunctional selection screen.

“Decoy Protein” Approach

An alternative approach to mitigate the observed toxicity in thedevelopment of a stable expression cell line while enabling theisolation of highly active amber suppressor cells, involves theintroduction of a surrogate gene containing an amber codon expressed athigh levels, which expression is driven by an inducible promoter toenable the down regulation of its expression during the expression ofthe target gene. This has the advantage that stable cell linesexpressing the PylRS/tRNApyl orthogonal machinery can be generated andused to modify multiple targets. To do this a eukaryotic expression hostcell such as CHO cells are transfected with a gene intended forexpression and containing one or more amber stop codons, such as but notlimited to GFP, eGFP, red fluorescent protein,glutathione-S-transferase, b-microglobulin, or B-galactoside cloned intoa mammalian expression vector preferably containing an induciblepromoter such as the Tet-On 3G (Clonthech), T-Rex (Life Technologies),ecdysone-inducible, or steroid-inducible promoters. Transfected cellsare selected by virtue of their resistance to, and ability to grow inmedium containing an appropriate antibiotic. Surviving cells are clonedby transferring 1-50 cells to each well of a 96-well plate and allowedto populate the well. Wells will then be assayed by ELISA assays, toidentify wells containing high titers of truncated protein. A highlyexpressed surrogate protein containing one or more amber codons willfunction as an amber sink to absorb amber suppression activity andprotect the cell from the deleterious effect of amber suppression.Functional elements such as U6-tRNA cassettes and pylRS genes, for theintroduction of nnAAs, are introduced into the host cell and selectedeither sequentially or concurrently. In one example, cells showing highexpression of the surrogate target gene are transfected with pMOAV2 orpMOAV2puro, containing genes for pylRS and pyltRNA. Transfected cellsare selected in DMEM containing 2 mM glutamax, 1 mM sodium pyruvate, 6mM glutamine, 1× non essential amino acids (Gibco CAT#11140-050), 10%fetal bovine serum, and 0.5 mg/mL hygromycin or 7.5 ug/ml puromycin.Surviving cells are propagated and 1-50 cells from this populationseeded into each well of 96 well plates. Once the cells have expandedand colonies form, cells are exposed to nnAA at 2 mM and functionallyassayed using ELISA assays to identify clones with amber suppressionefficiencies greater than 40% or 50% or preferably 40-60% or greaterthan 80 or 90% using a reporter protein (eGFPY40) or the surrogatetarget protein. High functioning clones are isolated by limitingdilution cloning or cell sorting. Genes encoding protein therapeuticswill then be introduced into these cells and selected. High expressingclones are isolated and identified by ELISA assays. High expressingclones will then be screened for amber suppression efficacy. The ratioof truncated to full length protein in each well is determined and wellsshowing high amber suppression activity, clones showing ambersuppression levels of at least 40% or 50%, but preferably 40-60% or80-90% or greater of the full length protein containing nnAA arepropagated.

If necessary additional tRNA genes will be introduced into theseselected pools of cells to further improve the efficacy of ambersuppression as just discussed above.

“Repressible tRNA” Approach

An alternative strategy has been engineered to regulate the tRNAexpression levels and mitigate tRNA associated cytotoxicity. To do this,promoter elements such as U6 or H1 necessary for tRNA expression aremodified to include sequence elements that enable the suppression ofgene expression such as the TetO repressor elements. This enables thedownregulation of tRNA expression during growth phase and the inductionof tRNA expression during expression of the target genes.

“Decoy Amino Acid” Approach

An alternative strategy to regulate the effects of background ambersuppression has been engineered by introducing an amino acid analoguerecognized by pylRS, and activated to the tRNApyl, but modified so as tonot allow peptide bond formation. The activation of this decoy aminoacid onto the tRNApyl will effectively compete with native amino acidactivation by host RSs or pylRS and generate a cellular pool of decoyamino acid activated tRNA. This pool will also compete with mis-acylatedtRNApyl for amber codons. The pool of decoy amino acid activated tRNAwill therefore allow for normal termination of protein synthesis atamber stop codons. During the course of platform cell line construction,cells such as DG44 CHO cells are grown in medium containing the decoyamino acid and the genes encoding tRNA and pylRS stably integrated intothese cells. Transfected cells are selected by growth in mediumcontaining appropriate antibiotics and surviving cells expanded. Thispool is transiently transfected with a vector encoding eGFPY40 and cellsgrown in medium containing a nnAA that allows peptide bond formationenabling amber codon readthrough, and lacking the decoy amino acid. Highfunctioning cells will then be identified by virtue of expression levelsof the eGFPY40 reporter and cells isolated using flow cytometry using aBD FACS Aria II. Sorted cells are expanded and the efficacy of ambersuppression in this sorted pool gauged using available reporters (e.g.eGFPY40 or FGF21-131amb). Iterative additions of tRNA or pylRS genes andselection using flow cytometry can be performed to enhance theefficiency of amber suppression if necessary. Platform cells will thenbe transfected with a target gene such as an IgG directed to a desirableantigen, containing an amber codon, in a vector containing the DHFR genesuch as the Optivec plasmid (Life Technologies) or a plasmid containingthe Glutamine Synthetase gene (Lonza) to allow for gene expressionselection. Cells expressing high levels of the truncated protein aregrown under appropriate selection, methotrexate or methioninesulphoximine respectively, to select for high expressing cells. Clonesare isolated using limiting cell dilutions and cells capable ofefficient amber suppression and high expression yields are identifiedusing ELISA assays.

Example 4: Modification of Target Proteins to Enable nnAA Incorporation

An amber codon was introduced into the ORF of the green fluorescenceprotein-blasticidin fusion in the vector pTracer His EF/HISA (LifeTechnologies) to generate the GFPY40 reporter construct. Briefly,site-directed mutagenesis was used to change the a single nucleotide atposition +120 (where +1 is the A of the start codon) of the GFP ORF froma cytosine to a guanine, and thereby generating an in-frame amber stopcodon.

FGF21 ORF was generated by gene synthesis using overlapping oligomersand PCR to regenerate the sequence for human FGF21 as shown in SEQ ID61,nucleotide sequence; SEQ ID62 amino acid sequence) containing anadditional amino terminal 3× Flag tag (encoding dykdhdgdykdhdidykddddks)(3×FLAG-FGF21, SEQ ID 80). The construct was cloned into the pJ201shuttle vector and transferred by restriction enzyme digestion usingHinDIII and XhoI and ligation to pCEP4 (Life Technologies). Theresulting construct placed the ORF of FGF21 downstream and under controlof a CMV promoter for expression in mammalian cells. Amber codons wereintroduced by site directed mutagenesis at positions F12 (SEQ ID 66),L66 (SEQ ID68), P90 (SEQ ID70), R131 (SEQ ID64), and P140 (SEQ ID71) ofthe FGF21 ORF. A two step PCR amplification scheme was used to replacethe 3×FLAG tag with a 6×His tag using overlapping oligomers. Briefly,two PCR reactions were set up, one to amplify the CMV promoter and asecond to amplify the FGF21 ORF and in frame with a 5′ 6×His tag.Flanking oligomers were then used in a third PCR reaction to join theCMV promoter to the 6×His-FGF21 construct. The product was cloned byGateway into a pDONR 221 P4r-P3r vector to generate both 6×HIS-FGF21 wtand 6×HIS-FGF21 R131.

An antibody directed against IL-6 was modified to enable the integrationof a nnAA and its subsequent conjugation. To generate this molecule thevariable regions of a rabbit antibody directed against the humancytokine IL-6 were grafted onto a human frameworks by PCR amplification(See WO12032181) and cloning into the vectors pFUSE-CHIg-hG1 (heavychain) and pFUSE-CHLIg-hK (Light chain)(Invivogen) to generate arabbit-human hybrid. Additional mutations were also incorporatedadjacent to the IL-6 CDRs to humanize the antibody. The resulting vectorpairs pFuse-28D2gamma and pFUSE-28D2kappa and served for cotransfectionand expression of the anti-IL-6 IgG by transient transfections. Thesites for nnAA incorporation were generated by introducing an ambercodon at the desired sites by site-directed mutagenesis and mutantsscreened by sequencing. This resulted in a heavy chain clone containingan amber codon at sites 274 (pFuse-28D2gamma_K274am) (SEQ ID 41). Cotransfection of the heavy chain constructs and the light chainconstructs allows expression of the anti-IL-6 antibody. An integratingconstruct containing the anti-IL-6 IgG heavy chain (containing an amberat position K274) was cloned into pOptivec by TOPO cloning. The Lightchain expression construct, including its promoter and poly A sequencewas amplified by PCR and a single copy of the tRNA were joined by twostep PCR method using overlapping oligomers and cloned into availablesites into the pOptivec plasmid containing the heavy chain(pOtivec-28D2-GKt). Transient expression and stable expression of theseantibodies was performed to integrate lysine-azide, ALOC,propargyl-lysine and lysine-chloride nnAAs.

Expression and Purification

For all experiments protein was isolated from stable cell lines (Example1). Alternatively, transiently transfected cell lines were utilized CHOor HEK293 cells were plated to approximately 90% confluence and grown at37 C. The following day, the plated cells were incubated with theappropriate DNA previously treated with a lipophilic reagent(Lipofectamine 2000, 293 fectin (invitrogen), according to the specificmanufacturer's instructions. Following 2-5 days of growth in thepresence of nnAA, ALOC, Lys-azide, propargyl Lysine or Lys-chloride) thegrowth medium was harvested and either used directly or the expressedproteins purified by an appropriate method. For expression of IgG, cellswere grown in medium containing low IgG fetal bovine serum. Stablytransfected cell lines were grown adherently in flasks to 90% confluenceand exposed to nnAA (selected from the following: ALOC(Nε-Allyloxycarbonyl-L-Lysine), lys azide, propargyl lysine,(2S)-2-amino-6-{[(2-azidoethyl)carbamoyl]oxy}hexanoic acid (FormulaVI.1)) for 5-7 days, and the growth medium harvested and either useddirectly or the expressed proteins purified by an appropriate method.For expression of IgG, cells were grown in medium containing low IgGfetal bovine serum.

Expressed IgGs, scFvs, or FGF21 described here were purified from growthmedium following stable or transient expression of eukaryotic cells. Ineach case 0.1 volumes of 10×PBS was added to the expression supernatantto equilibrate the salts and pH of the sample. For purification of 6×Histagged proteins, the supernatant was dialysed at 4° C. for 16 to PBS.Protein was bound to Nickle-NTA beads by batch binding or gravity flowand washed extensively with wash buffer (recipe). Bound material waseluted with (50 mM sodium phosphate pH7.4, 300 mM NaCl, 250-500 mMimidazole). Fractions containing the target protein were identified bySDS-PAGE and coomassie staining. Peak fractions were pooled and dialysedagainst PBS prior to further use.

IgGs were purified by protein A affinity chromatography. Briefly,expression supernatants were supplemented with 0.1 volumes of 10×PBS andpassaged through a 1 mL or 5 mL Protein A sepharose Fast Flow column(GE). Bound material was washed with 5-10 column volumes of PBS andeluted with 3-5 volumes of 0.1 M glycine pH3.0. Fractions weresubsequently neutralized by the addition of 0.05 volumes of 20×PBS toachieve a neutral pH. Elution fractions were analysed by SDS-PAGE andcoomassie staining and peak protein fractions pooled and dialysed to PBSat 4° C. for 16 hours.

This method was used to prepare: Anti-IL-6-LysAzide274h, FGF21 modifiedto include the NNAA (S)-2-amino-6((prop-2-ynyloxy)carbonylamino)hexanoicacid (Lys-Alkyne) at position 131

Example 5: Conjugation of nnAA-Containing Proteins

PEGylation of Anti-IL-6 Antibody with NNAA Lys-Azide Incorporated atPosition 274 of Heavy Chain with 20KPEG Terminal Alkyne(Anti-IL-6-LysAzide274h). (See FIG. 55)

In a 8×30 mm glass vial with small magnetic stirrer was placed adichloromethane solution of TBTA (80 mM, 3.75 mL), the solvent wasevaporated by gently blowing nitrogen over the tube. To this was added aphosphate buffer (125 mM, pH=7.4, 53 uL) and an aqueous solution of20KPEG alkyne (3 mM, 33 uL). A solution of the Anti-IL-6-LysAzide274hwas added (0.4 mg/mL, 6.26 uL) followed by a solution of cysteine (100mM, 2 uL) and copper sulfate (80 mM, 1.9 uL). The vial was blanketedwith argon, capped and mixed gently for 4h.

A portion of the reaction mixture was removed (15 uL) and mixed withnon-reducing gel loading buffer (4×, NuPage, Invitrogen, 7.5 uL). Theentire volume was loaded onto a SDS-PAGE gel for analysis (FIG. 5A):SDS-PAGE indicated the copper conditions afforded a mixture ofmonoPEGylated and bis-PEGylated antibody species. PDSi densitometryindicated the monoPEGylated species in approximately 1:1 ratio(mono=47%, bis=53%). The antibody with no azides failed to react undersimilar conditions, speaking to the specificity for the azide.

PEGylation of Anti-IL-6 Antibody with NNAA Lys-Azide Incorporated atPosition 274 of Heavy Chain (Anti-IL-6-LysAzide274h) with 20KPEGCYCLOOCTYNE (Bicyclo[6.1.0]Non-4-Yne-Linked PEG). (See FIG. 56)

In a 8×30 mm glass vial with small magnetic stirrer was placed phosphatebuffer (125 mM, pH=7.4, 60 uL) and an aqueous solution of 20KPEGcyclooctyne (bicyclicnonyne) (3 mM, 33 uL). A solution of theAnti-IL-6-LysAzide274h was added (0.4 mg/mL, 6.26 uL) and the vial wascapped and mixed gently for 4h.

A portion of the reaction mixture was removed (15 uL) and mixed withnon-reducing gel loading buffer (4×, NuPage, Invitrogen, 7.5 uL). Theentire volume was loaded onto a SDS-PAGE gel for analysis.

SDS-PAGE gel analysis, (FIG. 5B: Lane 1: (Anti-IL-6-LysAzide274h)treated with 20KPEG cyclooctyne, Lane 2: Antibody with no azides treatedwith 20K PEG cyclooctyne, Lane 3: Antibody untreated), indicated amixture of monoPEGylated and bis-PEGylated antibody species.Densitometry of the resulting gel indicated a mixture of monoPEGylated(10%) and bis-PEGylated antibody (76%), with the starting materialconsumed. The azide containing antibody was the only species to react.

To test the activity of antibodies directed against IL-6 and determinewhether the modification of this antibody altered the binding propertiesof this antibody we established an in vitro IL-6 neutralization assay.To do this, IL-6 dependent murine B-cell hybridoma cells (B9) wereseeded into 96 well plates in medium containing 50 pg/mL of IL-6.Different concentrations of an anti-IL-6 antibody and controls wereadded to a series of wells and grown for 3 days at 37° C. The viabilityof the cells was then determined using alamar blue a colorimetric assaythat allows to quantitatively measure the health of cells. Briefly, 25uL of reagent is added to each well and cells allowed to continuegrowing for 8-16 hr. After the incubation the panels are readspectrophotometrically at 570 nm and 600 nm wavelength. The measuresabsorbance is plotted versus the corresponding antibody concentrations.The data indicates that the site specific pegylation of the anti-IL-6antibody does not decrease the functional characteristics of theantibody as compared to naked unmodified antibody.

IL-6 inhibition assay (FIG. 8C). To test the activity of antibodiesdirected against IL-6 an IL-6 neutralization assay was used. IL-6dependent B9 cells were seeded into 96 well plates in medium containing50 pg/mL of IL-6. Different concentrations of a anti-IL-6 antibody andcontrols were added to a series of wells and grown for 3 days at 37° C.The viability of the cells was determined using alamar blue. Briefly, 25uL of reagent is added to each well and cells allowed to continuegrowing for 8-16 hr. After the incubation the panels are readspectrophotometrically at 570 nm and 600 nm wavelength. The measuresabsorbance is plotted versus the corresponding antibody concentrations.

Preparation of an Anti-IL-6 (Antibody)-Anti IL-23 (scFv-PEG) Bispecific.(See FIG. 57)

The generation of an scFv directed against the human cytokine IL23 wasgenerated using an E. coli expression system that enables site specificincorporation of an azido-homo alanine at desired sites. Briefly, amethionine auxotrophic strain of E. coli (B834) was transformed with anexpression plasmid encoding the scFv to hIL23 (WO2012/032181,incorporated herein by reference). The scFv gene encodes only twomethionines, the initiator methionine that is cleavedpost-translationally and a methionine at the c-terminus of the molecule.The transformed cells were fermented in rich medium and the cultureallowed to grow reach a growth plateau. At this point the cells wereinduced with IPTG to derepress expression of the scFv and the mediumsupplemented with AHA. The cells were then allowed to grow for anadditional four hours and the bacterial cells harvested bycentrifugation. The expressed scFv was purified from inclusion bodiesand folded in vitro. The expressed scFv contains an azide containingamino acid that allows for conjugation with alkyne containing moieties.To generate a bi-specific antibody construct the Anti-IL-6-LysAzide274hwas ligated to the anti-IL23 scFv with bis-alkyne PEG moiety(WO2012/032181, incorporated herein by reference). To do this a 8×30 mmglass vial with small magnetic stirrer was placed phosphate buffer (20mM, pH=7.4, 80 uL). A solution of the Anti-IL-6-LysAzide274h was added(0.4 mg/mL, 6.3 uL), followed by a solution of the anti-IL23 scFvpreviously conjugated to a −20KPEG alkyne (1 mg/mL, 2.1 uL). To thissolution was added a solution of BME (100 mM, 3 uL) and a solution ofcopper sulfate (80 mM, 2.81 uL). The mixture was allowed to stir for 4h.

A portion of the reaction mixture was removed (15 uL) and mixed withnon-reducing gel loading buffer (4×, NuPage, Invitrogen, 7.5 uL). Theentire volume was loaded onto a SDS-PAGE gel for analysis. A very smallamount of the bispecific was produced as evidenced by the new highermolecular band above the antibody band (FIG. 6A).

For reducing gels, a portion of the reaction mixture was removed (15 uL)and mixed with reducing gel loading buffer (4×, NuPage, Invitrogen, 30%BME, 7.5 uL). The entire volume was loaded onto a SDS-PAGE gel foranalysis. A very small amount of the bispecific was produced asevidenced by the new higher molecular band above the heavy chain band,consistent with the anticipated MW change (FIG. 6B).

PEGylation of FGF-21 (Modified to Include NNAA Lys-Alkyne) with 20KLinear PEG Bis Azide

For all experiments standard transfection conditions were use. Briefly,a clone of 293 cells previously selected for stable pylRS expressionwere plated to approximately 90% confluence and grown at 37° C. for 16h.The following day, the plated cells were treated with the 6×HIS-FGF21R131 previously combined with a lipophilic reagent (Lipofectamine 2000,293 fectin (invitrogen), according to the specific manufacturer'sinstructions. Cells were then grown in DMEMcomplete (DMEM, 2 mMglutamax, 1 mM sodium pyruvate, 6 mM glutamine, 1× non essential aminoacids, 10% fetal calf serum) medium containing 2 mM propargyl-lysinennAA, for 5-7 days. The growth medium was harvested and FGF21 purifiedby affinity chromatography on a 5 mL prepacked nickel-NTA column (GE).

The expressed 6×HIS-FGF21 was purified from the growth medium. Here, 0.1volumes of 10×PBS was added to the expression supernatant to equilibratethe salts and pH of the sample. And the medium was dialysed at 4° C. for16 to PBS. The expressed FGF21 was bound to a Nickel-NTA beads (GE) bybatch binding or gravity flow and washed extensively with wash buffer(50 mM sodium phosphate pH7.4, 300 mM NaCl, 20 mM imidazole). Boundmaterial was eluted with NiNTA elution buffer (50 mM sodium phosphatepH7.4, 300 mM NaCl, 250-500 mM imidazole). Fractions containing thetarget protein were identified by SDS-PAGE and coomassie staining. Peakfractions were pooled and dialysed against PBS prior to further use.

In a 20 mL vial with magnetic stirrer was placed a solution of FGF21modified to include the NNAA(S)-2-amino-6((prop-2-ynyloxy)carbonylamino)hexanoic acid (Lys-Alkyne)at position 131 (SEQ ID64) (20 ug/mL, 0.001 mM, 5000 uL). To this wasadded a solution of 20K linear PEG bis-azide (60 mg/mL, 1.67 mL). Asolution of SDS (20%, 250 uL) and a solution of DTT (250 mM, 60 uL) wereadded. A DMSO solution of TBTA (80 mM, 7.96 uL) and an aqueous solutionof copper sulfate (80 mM, 94 uL). The vial was capped and the reactionwas allowed to stir overnight. The mixture was centrifuged (10000 g, 15min) and the supernatant retained. The reaction mixture was assessed bySDS-PAGE and a clear molecular weight shift was observed consistent withthe conjugation of 20 kDa PEG to the polypeptide (FIG. 7A).

To assess the potency of the PEGylated FGF21 constructs obese, db/dbmice were treated daily with 0.25 mg/Kg FGF21 or 20K PEG-FGF21 andglucose levels measured in fed mice after three treatments using ahandheld glucose monitor, (FIG. 7B).

The homozygous db/db mouse (B6.BKS(D)-Leprdb/j, Jackson labs) is a wellcharacterized animal model for diabetes and becomes obese after 3-4weeks of age. The animals also display elevated plasma insulin, bloodsugar, and delayed wound healing. In this study, 7-8 week old, maledb/db mice were fed Lab Diet 5053 Rodent Diet 20 ad libitum. Mice wereacclimatized for seven days and subsequently administered PEGylatedFGF21, unmodified FGF21 and vehicle (PBS) subcutaneously daily foreleven days.

Each mouse was administered 0.25 mg/Kg of PEG-FGF21 or unmodified FGF21daily for three days. Fed glucose blood levels were determined by tailclip bleeding one hour after compound administration on day 3, andglucose levels measured by a handheld glucose meter (Bayer). The datashows that the PEGylation of FGF21 at amino acid residue 131 has thesame potency of wild type FGF21 and shows improved glucose levelmaintenance as compared to placebo controls.

Example 6: Preparation of Amino Acids and Decoy Amino Acids

2-{[(benzyloxy)carbonyl]amino}-6-{[(prop-2-en-1-yloxy)carbonyl]amino}hexanoicacid (Formula VIIA.4, BaChem),2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-6-{[(prop-2-en-1-yloxy)carbonyl]amino}hexanoic acid (Formula VIIA.5, BaChem),6-{[(tert-butoxy)carbonyl]amino}hexanoic acid (Formula VIIB.4) werepurchased from commercial vendors.

Preparation of Decoy nnAAs of Formula VIIA

Formula VII analogs are readily prepared by acylating the α-amino groupof the starting amino acid with an activated electrophile. This is doneby treatment of the starting material with an acid chloride, activatedester, anhydride or sulfonyl chloride. The product can then be utilizedfor cell line development.

Preparation of Decoy Amino Acids of Formula VIIB:

Preparation of 6-{[(prop-2-en-1-yloxy)carbonyl]amino}hexanoic acid(Formula VIIB.1)

In a 20 mL vial with magnetic stirrer was placed 6-aminocaproic acid(280 mg), sodium hydroxide (1M, 5.3 mL) and dioxane (2 mL). Allylchloroformate (228 uL) was added and mixture stirred for 3h. The mixturewas treated with 1M citric acid until pH was acidic. The mixture wasextracted with ethyl acetate (×3) and the organic layers retained. Theorganic layers were combined, dried with sodium sulfate, filtered andconcentrated. Analytical MS: m/z (ES+) calculated 215.1 (M+H)+. found216.1.

Preparation of 6-{[(2-chloroethoxy)carbonyl]amino}hexanoic acid (FormulaVIIB.3)

In a 20 mL vial with magnetic stirrer was placed 6-aminocaproic acid(1180 mg), sodium hydroxide (1M, 22.5 mL) and dioxane (23 mL).2-chloroethyl chloroformate (932 uL) was added and mixture stirred for3h. The mixture was treated with excess 1M citric acid until pH wasacidic. The mixture was extracted with ethyl acetate (×3) and organiclayers retained. The organic layers were combined, dried with sodiumsulfate, filtered and concentrated. Analytical MS: m/z (ES+) calculated237.1 (M+H)+. found 238.1.

Preparation of 6-{[(2-azidoethoxy)carbonyl]amino}hexanoic acid (FormulaVIIB.6)

In a 20 mL vial with magnetic stirrer was placed 6-{[(2-chloroethoxy)carbonyl]amino} hexanoic acid (250 mg) and DMSO (5 mL). Sodium azide wasadded (2-chloroethyl chloroformate (70 mg) was heated to 60 C andstirred for 20h. The mixture was diluted with water (5 mL) and pouredonto 1M citric acid (10 mL). The mixture was extracted with ethylacetate (×3) and organic layers retained. The organic layers werecombined and washed with 5% lithium chloride. The organic layer wasdried with sodium sulfate, filtered and concentrated. Analytical MS: m/z(ES+) calculated 244.1 (M+H)+. found 245.2.

Preparation of 6-{[(prop-2-yn-1-yloxy)carbonyl]amino}hexanoic acid(Formula VIIB.5)

In a 20 mL vial with magnetic stirrer was placed 6-aminocaproic acid(220 mg), sodium hydroxide (1M, 4.2 mL) and dioxane (2 mL). Propargylchloroformate (163 uL) was added and mixture stirred for 3h. The mixturewas treated with excess 1M citric acid until pH was acidic. The mixturewas extracted with ethyl acetate (×3) and organic layers retained. Theorganic layers were combined, dried with sodium sulfate, filtered andconcentrated. Analytical MS: m/z (ES+) calculated 213.1 (M+H)+. found214.1.

Preparation of 5-{[(prop-2-en-1-yloxy)carbonyl]amino}pentanoic acid(Formula VIIB.2)

In a 500 mL round bottomed flask with magnetic stirrer was placed5-aminovaleric acid (15.0 g), water (100 mL) and 2N sodium carbonate (40mL). Allyl chloroformate (8.2 mL) in dioxane (100 mL) was added dropwiseand the final mixture stirred for 3h. The mixture was acidified with 2NHCl (˜50 mL). The mixture was extracted with ethyl acetate (4×100 mL)and the organic layers retained. The organic layers were combined, driedwith sodium sulfate, filtered and concentrated. Analytical MS: m/z (ES+)calculated 201.1 (M+H)+. found 202.1.

Example 7. Preparation of Formula V and VI Analogs Preparation ofPreparation of (S)-2-amino-6((2-oxo-2-phenylacetamide)hexanoic acid(Formula V.6)

In a 50 mL round bottomed flask with magnetic stirrer was dissolvedpyruvic acid (3.5 g, 23.3 mmol) in a 2:1 mixture of dichloromethane andDMF (20 mL). To this mixture was added DCC (5.7 g, 27.6 mmol) and NHS(3.2 g, 27.6 mmol). The mixture was heated to 50 C for 30 min withstirring. The solution was allowed to cool and then added through afilter to a suspension of N-Boc-Lysine (5.2 g, 21.2 mmol) in DMF (20 mL)in a separate 100 mL round bottomed flask with magnetic stirrer.Triethyl amine (8.8 mL, 63.6 mmol) was added after addition of theactivated ester, and the mixture was stirred overnight. The mixture waspartitioned between ethyl acetate and citric acid. The layers wereseparated and the aqueous layer was extracted 4 times with ethylacetate. The organic layers were combined, dried over sodium sulfate andconcentrated. The resulting residue was further purified by flashchromatography to afford the final N-Boc lysine derivative as an oil.

In a 100 mL roundbottomed flask was placed the keto-N-Boc lysinederivative (4 g, 10.6 mmol) in acetonitrile (50 mL). To this was added asolution of hydrochloric acid (15 mL, 4N in dioxane). The solution wasstirred for 2h and concentrated. Final purification by flashchromatography afforded the target amino acid. Analytical MS: m/z (ES+)calculated 278.1 (M+H)+. found 279.1.

Preparation of (2S)-2-amino-6-(2-azidoacetamido)hexanoic acid (FormulaV.8)

In a 25 mL roundbottomed flask was placed N-Boc-Lysine (500 mg, 2.0mmol) suspended in dioxane (5 mL). Saturated NaHCO₃ was added (2 mL) andthe solution was cooled to 0° C. Bromoacetyl chloride (169 uL, 2.0 mmol)in dioxane (2 mL) was added slowly. The solution was allowed to stir at0 C for 1 h and then at room temperature for 4h. The solution wastransferred to a extraction funnel and partitioned between water andether. The organic layer was removed and the aqueous layer made acidic(pH=2) with citric acid. The aqueous layer was extracted with ethylacetate (3×50 mL), the organic layers combined and dried over sodiumsulfate, filtered and concentrated. The resulting residue was carriedforward into the next step.

In a 50 mL round bottomed flask was placed the crudeN-Boc-ε-2-bromoacetyl-lysine (740 mg, 2.0 mmol) in dioxane (10 mL). Tothis was added a solution of sodium azide (10 mL, 1M). The solution wasstirred at 60° C. overnight. The mixture was partitioned between citricacid (1M, 50 mL) and ethyl acetate (100 mL). The organic layer wasretained, and the aqueous layer extracted 3 additional times. Theorganic layers were combined, dried over sodium sulfate and concentratedto an oil.

The crude N-Boc-ε-2-azido-acetyl-lysine was dissolved in acetonitrile(10 mL) and TFA (2 mL) was added. The mixture was stirred for 2h andthen concentrated. The solution was treated with toluene (10 mL) andconcentrated (2×) and acetonitrile (10 mL) and concentrated (2×). Theresidue was dried overnight under vacuum. The residue was taken up inMeOH and precipitated with methyl-t-butyl ether. The viscous oil wasisolated by centrifugation, the supernatant was disposed. Analytical MS:m/z (ES+) calculated 229.1 (M+H)+. found 230.1.

Preparation of (2S)-2-amino-6-(pent-4-enamido)hexanoic acid (FormulaV.5)

In a 25 mL roundbottomed flask was placed N-Boc-Lysine (500 mg, 2.0mmol) suspended in dioxane (10 mL). 1M K₂CO₃ was added (5 mL) and thesolution was cooled to 0 C. 4-pentenoyl chloride (224 uL, 2.0 mmol) indioxane (2 mL) was added slowly. The solution was allowed to stir at 0 Cfor 1 h and then at room temperature for 4h. The solution wastransferred to a extraction funnel and partitioned between water andether. The organic layer was removed and the aqueous layer made acidic(pH=2) with citric acid. The aqueous layer was extracted with ethylacetate (3×50 mL), the organic layers combined and dried over sodiumsulfate, filtered and concentrated. The resulting residue was carriedforward into the next step.

The crude N-Boc-ε-N-4-pentenoyl amide-lysine was placed in a 50 mL roundbottomed flask with acetonitrile (5 mL) and TFA (2 mL) and magneticallystirred for 2h. The mixture was concentrated. The solution was treatedwith toluene (10 mL) and concentrated (2×) and acetonitrile (10 mL) andconcentrated (2×). The residue was dried overnight under vacuum. Theresidue was taken up in MeOH and precipitated with methyl-t-butyl ether.The viscous oil was isolated by centrifugation, the supernatant wasdisposed. Analytical MS: m/z (ES+) calculated 229.2 (M+H)+. found 229.1.

Preparation of Hydroxy-Norleucine Derivatives (Formula VI.1 and FormulaVI.2)

In a 100 mL roundbottomed flask with magnetic stirring was placedN-Boc-Hydroxyl Norleucine (1 g, 4.4 mmol) and acetonitrile (50 mL). Themixture was cooled to 0° C. and p-nitrophenylchloroformate (979 mg, 4.9mmol) and Pyridine (2 mL) was added and the mixture stirred overnight.The mixture was concentrated and purified by flash chromatography.(Silica, DCM/MeOH gradient).

In a 100 mL roundbottomed flask with magnetic stirring was placed2-N-Boc-ethylbromide (1 g, 4.4 mmol) in 25 mL of dioxane. To this wasadded a solution of sodium azide (1M, 22.2 mmol). The solution wasstirred at 60° C. overnight. The mixture was partitioned between waterand ethyl acetate. The ethyl acetate layer was retained and the aqueouslayer was extracted with ethyl acetate three additional times. Theorganic layers were combined, dried over sodium sulfate and concentratedto an oil.

The oil was taken up in acetonitrile (35 mL) and HCL in dioxane wasadded (4M, 10 mL). The mixture was stirred for two hours andconcentrated under vacuum.

Preparation of (2S)-2-amino-6-{[(2-azidoethyl)carbamoyl]oxy}hexanoicacid (Formula VI.1)

In a 50 mL round bottomed flask was placed the N-Boc-Norleucinep-nitrophenyl carbonate (503 mg, 1.2 mmoL) in dioxane (10 mL). To thiswas added a solution of the amino-azide (105 mg, 1.2 mmol) in dioxane (5mL) and pyridine (1 mL). The solution was stirred overnight. The mixturewas partitioned between ethyl acetate and 500 mM citric acid. The ethylacetate layer was retained and the aqueous layer was extracted withethyl acetate three additional times. The organic layers were combined,dried over sodium sulfate and concentrated to an oil. The oil wasfurther purified by flash chromatography.

The isolated Boc-protected amino acid was taken up in acetonitrile (15mL) and treated with HCl in dioxane (4M, 5 mL). The mixture was stirredfor two hours and concentrated under vacuum.

Preparation of (2S)-2-amino-6-{[(prop-2-yn-1-yl)carbamoyl]oxy}hexanoicacid (Formula VI.2)

In a 50 mL round bottomed flask was placed the N-Boc-Norleucinep-nitrophenyl carbonate (337 mg, 0.8 mmoL) in dioxane (10 mL). To thiswas added a solution of the amino-azide (135 mg, 2.4 mmol) in dioxane (5mL). The solution was stirred overnight. The mixture was partitionedbetween ethyl acetate and 500 mM citric acid. The ethyl acetate layerwas retained and the aqueous layer was extracted with ethyl acetatethree additional times. The organic layers were combined, dried oversodium sulfate and concentrated to an oil. The oil was further purifiedby flash chromatography.

The isolated Boc-protected amino acid was taken up in acetonitrile (15mL) and treated with HCl in dioxane (4M, 5 mL). The mixture was stirredfor two hours and concentrated under vacuum.

Example 8: Anti-Her2-Toxin Conjugation

The anti-Her2 antibody was obtained as follows.

The variable regions of the mouse antibody 4D5 directed to theextracellular domain of Her2 was generated by gene synthesis usingoverlapping oligomers and cloned into a shuttle vector. The variableregions were then grafted onto the human frameworks encoded bypFUSE-CHIg-hG1 and pFUSE-CHLIg-hK (Invivogen) to generate a mouse-humanhybrid. Amber codons were introduced into the heavy chain (gamma) atpositions 274 and 359 (SEQ ID 47 and SEQ ID 49 respectively) and thelight chain (Kappa) at positions 70 and 81 (SEQ ID 53 and SEQ ID55respectively) by site directed mutagenesis. Clones containing the ambercodon were identified by DNA sequencing. To generate an integratingconstruct in pOptivec for this IgG, the promoters and ORF for the heavychain was amplified by PCR and cloned by restriction enzyme digestionand ligation into pOptivec. The light chain and a single copy of thetRNA were joined by two step PCR method using overlapping oligomers andcloned into available sites into the pOptivec plasmid containing theheavy chain.

Expression and Purification

An antibody directed against HER2 was generated by gene synthesis of theHerceptin CDRs and the IgG1 framework modified to enable the integrationof a nnAA at one or two sites and their subsequent conjugation. Themurine CDRs of Herceptin were cloned into pFUSE-CHIg-hG1 (heavy chain)and pFUSE-CHLIg-hK (Light chain)(Invivogen) to generate a humanizedantibody. The resulting vector pairs pFuse-4D5gamma and pFUSE-4D5kappaserved for cotransfection and expression of the wildtype anti-Her2 IgGby transient transfections (SEQ ID 45, SEQ ID 46, heavy chain; SEQ ID51, SEQ ID 52, light chain). The sites for nnAA incorporation weregenerated by introducing an amber codon at the desired sites bysite-directed mutagenesis and mutants screened by sequencing. Thisresulted in a heavy chain clone containing an amber codon at position274 (pFuse-4D5gamma_K274am) (SEQ ID 47). Amber sites were alsoconstructed in pFUSE-4D5kappa. First the termination codon was replacedfrom an amber codon to an ochre stop codon to generate the vectorpFUSE-4DSkappa_TAA. An amber codon at position D70 was introduced bysite directed mutagenesis (SEQ ID 53). By pairing these differentvectors antibodies containing a single nnAA or two nnAAs can begenerated.

Transient expression of target antibodies containing a nnAA wereperformed in HEK293 cells stably expressing pylRS. This cell line wasgenerated by transfection of a vector containing the pylRS gene in pCEP4(Life Technologies) and selection by growth in medium containinghygromycinB (DMEM (Life Technologies), 2 mM glutamax, 1 mM sodiumpyruvate, 6 mM glutamine, 1× non essential amino acids (GibcoCAT#11140-050), 10% fetal calf serum, and 0.2 mg/mL hygromycin).Surviving cells were cloned by limiting dilution and clonesdemonstrating high functional activity of the pylRS were expanded. Thiswas achieved by transiently transfecting the different clones with avector encoding tRNApyl and a reporter construct GFPY40 containing anamber codon at position Y40 in the presence of ALOC nnAA. Fluorescencelevels were quantified in these cells using an Accuri flow cytometer andhigh functioning clones isolated. Expression of the anti-Her2 antibodieswas performed using standard transfection conditions. Cells were platedto approximately 90% confluence and grown at 37 C. The following day,the plated cells were incubated with the appropriate DNA previouslytreated with a lipophilic reagent (Lipofectamine 2000, 293 fectin(invitrogen), according to the specific manufacturer's instructions.Following 2-5 days of growth in the presence of 1-2 mM Lys-azide, thegrowth medium was harvested and either used directly or the expressedproteins purified by an appropriate method. For expression of IgG, cellswere grown in medium containing low IgG fetal bovine serum. In each case0.1 volumes of 10×PBS was added to the expression supernatant toequilibrate the salts and pH of the sample and antibodies purified byprotein A affinity chromatography. Briefly, expression supernatants werepassaged through a 1 mL or 5 mL nProtein A sepharose Fast Flow column(GE). Bound material was washed with 5-10 column volumes of PBS andeluted with 3-5 volumes of 0.1 M glycine pH3.0. Fractions weresubsequently neutralized by the addition of 0.05 volumes of 20×PBS toachieve a neutral pH. Elution fractions were analysed by SDS-PAGE andcoomassie staining and peak protein fractions pooled and dialysed to PBSat 4° C. for 16 hours. IgGs containing a lys azide as a nnAA arereferred to as AzAb.

Preparation of Cytotoxin-Alkyne Derivatives

Preparation of MMAF-Alkyne Derivative.

Monomethyl auristatin F (MMAF) (6 mg, 8.2 umol) was placed in a smallvial, and DMSO (450 uL) was added. A solution of BCN carbonate in DMSO(82.6 ug/uL, 84 uL, 2.4 mg, 8.2 umol) was added to the MMAF solution.Triethylamine (2.5 uL, 18 umol) was added, the vial capped and thereaction stirred for 4h. Analytical MS: m/z (ES+) calculated 907.1(M+H)+. found 908.6.

Preparation of MMAF-Valine-Citruline-p-Amino-Benzoyl-Carbonate(VCP)-Cyclooctyne Derivative.

In a 4 mL vial with magnetic stirrer was placed MMAF (5 mg, 6.84 umol)and the dipeptide val-cit-PABC-Fmoc (5.24 mg, 6.84 umol). To thismixture was added a DMSO (350 uL). A DMSO solution ofethyl(hydroxyimino)cyanoacetate (40 mg/mL, 25 uL), and an aqueoussolution of potassium tert-butoxide (60 mg/mL, 25 uL) was added, thevial was capped and allowed to stir overnight. Analytical MS: m/z (ES+)calculated 1358 (M+H)+. found 1359.8. The crude mixture was useddirectly in the next step.

The crude MMAF-VCP-Fmoc was treated taken up in 400 uL ofdichloromethane and treated with 400 uL of diisopropylamine. The mixturewas stirred for 2h, transferred to a roundbottomed flask with methanoland concentrated. The material was treated with heptanes (2 mL) andconcentrated, the sequence was repeated with isopropanol to removeexcess diisopropylamine. The material was concentrated under high vacuumovernight and carried on to the next step. Analytical MS: m/z (ES+)calculated 1136.7 (M+H)+. found 1137.6.

The crude MMAF-VCP-NH2 was taken up in DMF (420 uL) and treated with asolution of ALKYNE carbonate (40 mg/mL, 50 uL) and triethylamine (2.8uL). The mixture was stirred for 8h at room temperature. Analytical MS:m/z (ES+) calculated 1312.8 (M+H)+. found 1313.7.

Preparation of Paclitaxel-Cyclooctyne Derivative.

Paclitaxel (500 mg, 590 umol) and glutaric anhydride were placed in a 50mL round bottomed flask with magnetic stir and pyridine (10 mL) wasadded. The solution was stirred overnight. The mixture was concentratedto an oil and purified by column chromatography on silica gel(hexane/acetone elution) affording the desired product. Analytical MS:m/z (ES+) calculated 968.0 (M+H)+. found 969.1.

A solution of taxol-glutaric acid conjugate in DMF (15.6 ug/uL, 321 uL,5 mg, 5.2 umol) was placed in a small vial with magnetic stir bar. Asolution of HATU coupling agent (46.1 ug/uL, 50 uL, 2.3 mg, 6.2 umol), asolution of cyclooctyne-amine (34 ug/uL, 50 uL, 1.7 mg, 5.2 umol) andtriethylamine (1.6 uL, 11.4 umol) were added in succession. The vial wascapped shut and stirred overnight. Analytical MS: m/z (ES+) calculated1273.6 (M+H)+. found 1274.4.

Preparation of a Doxorubicin-Cyclooctyne Derivative.

A doxorubicin solution (12.5 ug/uL, 320 uL, 4 mg, 7.4 umol) was placedin a small vial with small magnetic stir bar. A solution of cyclooctynecarbonate in DMSO (28.5 ug/uL, 63 uL, 1.8 mg, 7.4 umol) was added to thevial. Triethylamine (2.2 uL, 16 umol) was added, the vial capped and thereaction stirred for 4h. Analytical MS: m/z (ES+) calculated 719.7(M+H)+. found 720.5.

Conjugation of Anti-Her2 Antibody, nnAA Lys-Azide Incorporated atPosition 274 of Heavy Chain (Anti-Her2-LysAzide274h) withMMAF-Cyclooctyne Derivative. (See FIG. 58)

In a 200 uL PCR tube was placed a solution of phosphates (50 mM, pH=7.4,3 uL) and a solution of the anti-Her2 Antibody (Anti-Her2-LysAzide274h)(11.43 uL, 2.1 mg/mL). To this was added a DMSO solution of theMMAF-ALKYNE derivative (1.1 uL, 14.5 mMol), the tube was capped andvortexed. The mixture was allowed to stand for 4h. The reaction mixturewas then treated with a solution of azidohomoalanine (AHA, 250 mM in 1MHEPES, 6.4 uL), vortexed and allowed to stand for 60 min. The mixturewas then desalted through a ZEBA (Pierce) mini spin column to afford thefinal ADC solution (0.21 mg/mL)

A cell-based fluorescence assay was used to show that the 4D5 IgG andconjugated 4D5-MMAF bound and internalized into cells expressing Her2epitope. The breast cancer cell lines A345, or SKBR3 and EL4 and EL4cells, stably transfected with a construct for the expression of Her2,were grown in complete RPMI-1640 or DMEM. Cells were dissociated,counted and harvested by centrifugation. For each assay approximately200,000-500,000 cells were incubated with PBS containing 0.5% BSA for 1hour at RT. Cells were then treated with 1 ug of purified 4D5 IgG or 4D5IgG-MMAF conjugate in the presence of absence of 0.1% sodium azide for 1hour at 37° C. Cells were washed and incubated with an anti-HumanIgG-phycoerythrin conjugate for 1 hour at 37° C., washed with PBS, andresuspended in PBS or PBS containing 50% Trypan Blue and analysed byflow cytometry (Accuri).

Cell viability and cell death assays. The effect of the anti-Her2-MMAFconjugate on tumor cell viability was assessed using an MTS assay.Briefly, cells were plated onto 96 well plates (5000 cells per well ofSKBR3, MDA-MD, and MCF7) in 50 uL of RPMI 1640 lacking phenol red andcontaining 10% fetal bovine serum (FBS). Different concentrations of theantibody conjugates and controls were added in 50 uL of RPMI1640containing FBS to the cells for three days at 37° C. in a humidifiedenvironment of 5% CO2. Cell viability was analysed by the addition of 20uL of complete MTS (Pierce) and color allowed to develop for 1-3 hoursat 37° C. The absorbance of each well at 490 nm was recorded using aplate reader (Molecular Dynamics) (FIG. 8B)

Conjugation of Anti-Her2 Antibody (Anti-Her2-LysAzide274h) withMMAF-Valine-Citruline-p-Amino-Benzoyl-Carbonate-Cyclooctyne Derivative

In a 200 uL PCR tube was placed a solution of phosphates (50 mM, pH=7.4,3 uL) and a solution of the anti-Her2 Antibody (NNAA lys-azideincorporated at position 274 of heavy chain) (11.43 uL, 2.1 mg/mL). Tothis was added a DMSO solution of the MMAF-ALKYNE derivative (1.23 uL,13 mMol), the tube was capped and vortexed. The mixture was allowed tostand for 4h. The reaction mixture was then treated with a solution ofazidohomoalanine (AHA, 250 mM in 1M HEPES, 6.4 uL), vortexed and allowedto stand for 60 min. The mixture was then desalted through a ZEBA(Pierce) mini spin column to afford the final ADC solution (0.21 mg/mL)

Conjugation of Anti-Her2 Antibody (Anti-Her2-LysAzide274h) withPaclitaxel-Cyclooctyne Derivative

In a 200 uL PCR tube was placed a solution of phosphates (50 mM, pH=7.4,3 uL) and a solution of the anti-Her2 Antibody (Anti-Her2-LysAzide274h)(11.43 uL, 2.1 mg/mL). To this was added a DMSO solution of thePaclitaxel-ALKYNE derivative (1.24 uL, 12.9 mMol), the tube was cappedand vortexed. The mixture was allowed to stand for 4h. The reactionmixture was then treated with a solution of azidohomoalanine (AHA, 250mM in 1M HEPES, 6.4 uL), vortexed and allowed to stand for 60 min. Themixture was then desalted through a ZEBA (Pierce) mini spin column toafford the final ADC solution.

Example 9: PEGylation to Anti-Her2 Antibody (Anti-Her2-LysAzide274h)

PEGylation with 20K Linear PEG-Cyclooctyne to Anti-Her2 Antibody(Anti-Her2-LysAzide274h) (See FIG. 59)

In a 200 uL PCR tube was placed phosphate buffer (50 mM, pH=7.4, 1 uL).A solution of azide containing antibody (AzAb-2, 2.1 mg/mL, 1.07 uL) wasadded followed by a solution of 20KPEG cyclooctyne (60 mg/mL, 1.0 uL).The solution was mixed vigorously on a vortexer (Fisher). The tube wasplaced on a PCR tube centrifuge for a few seconds to place all liquidsinto the bottom of the tube. The mixture was allowed to stand for 4h.

The solution was diluted with water (7 uL) to bring the final volume to˜10 uL. The solution was then partitioned into 5 uL and added to 5 uL ofeither reducing or non-reducing gel loading buffer. The mixture wasmixed and heated to 95 C for 3 minutes. The samples were then loadedonto SDS-PAGE gels (4-20% Tris-Gly, Invitrogen). SDS-PAGE (reducing andnon-reducing) indicated that the PEGylation occurred with a high degreeof conversion with nearly all starting being converted to thebis-PEGylated species. Non reducing gel (FIG. 9A) lane 2: anti-Her2Antibody (NNAA lys-azide incorporated at position 274 of heavy chain)untreated, Lane 3: anti-Her2 Antibody (NNAA lys-azide incorporated atposition 274 of heavy chain) treated with 20KPEG linear PEG cyclooctyne.A clear molecular shift is observed in the PEG treated Azide-Antibody toa dominant single species, consistent with the anticipated withmolecular weight shift of inclusion of two PEG chains. PDSI indicated ahigh degree conversion (93% bis PEGylation, 6.9% mono-PEGylation, nostarting material). Reducing gel (FIG. 9B) Lane 2: anti-Her2 Antibody(NNAA lys-azide incorporated at position 274 of heavy chain), Lane 3:anti-Her2 Antibody (NNAA lys-azide incorporated at position 274 of heavychain) treated with 20KPEG linear PEG alkyne. A clear molecular weightshift of the heavy chain in the PEG treated antibody (Lane 3) wasobserved, speaking to the specificity for the azide containing heavychain and the degree of conversion with the PEG conjugation (96.7%,densitometry).

PEGylation with 20KPEGcyclooctyne to Anti-Her2 Antibody with nnAALys-Azide Incorporated at Position 274 of Heavy Chain and Position 70 ofLight Chain (Anti-Her2-LysAzide274h70l)

In a 200 uL PCR tube was placed a solution of theAnti-Her2-LysAzide274h70l antibody 0.5 mg/mL, 4.5 uL). A solution of20KPEG cyclooctyne (60 mg/mL, 1.0 uL) was added and the solution mixedvigorously on a vortexer (Fisher). The tube was placed on a PCR tubecentrifuge for a few seconds to place all liquids into the bottom of thetube. The mixture was allowed to stand for 18h.

The solution was diluted with water (4.5 uL) to bring the final volumeto ˜10 uL. The solution was then partitioned into 5 uL and added to 5 uLof either reducing or non-reducing gel loading buffer. The gel sampleswere mixed and heated to 95 C for 3 minutes. The samples were thenloaded onto SDS-PAGE gels (4-20% Tris-Gly, Invitrogen). SDS-PAGE(non-reducing) indicated that the PEGylation occurred with a high degreeof conversion with nearly all starting antibody converted to thetetra-PEGylated species. Non reducing gel (FIG. 10A lane 2: anti-Her2Antibody (Anti-Her2-LysAzide274h70l) untreated, Lane 3-5: All treatedwith 20K linear PEG cyclooctyne Lane 3: anti-Her2 Antibody(Anti-Her2-LysAzide274h), Lane 4: anti-Her2 Antibody(Anti-Her2-LysAzide274h), Lane 5: Herceptin (no azides) negativecontrol, Lane 6: anti-Her2 Antibody (Anti-Her2-LysAzide274h) untreatedand Lane 7: Herceptin untreated. A clear molecular weight shift isobserved from the single band of the untreated 4D5 AzAb-4 to thetetra-PEGylated species which is dominant. This tetra-PEGylated speciesis larger than the bis-PEGylated species (Lane 4). The non-reducing gelalso shows the specificity of the reaction for azide containingantibodies, with Herceptin, containing no azides, showing no reactivity.PDSI indicated a high degree conversion (86% bis PEGylation, 14tris-PEGylation, no starting material). Reducing gel (FIG. 10B) lane 2:anti-Her2 Antibody (Anti-Her2-LysAzide274h70l) untreated, Lane 3-5: Alltreated with 20K linear PEG cyclooctyne Lane 3: anti-Her2 Antibody(Anti-Her2-LysAzide274h70l), Lane 4: anti-Her2 Antibody(Anti-Her2-LysAzide274h), Lane 5: Herceptin (no azides) negativecontrol, Lane 6: anti-Her2 Antibody (Anti-Her2-LysAzide274h) untreatedand Lane 7: Herceptin untreated. The reducing gel shows that both theheavy and light chains (lane 3) underwent a clear molecular shift,consistent with the addition of a single 20KPEG chain to each subunit ofthe antibody. The bands are distinct, indicating the reaction took placeonly at the azide site and no additional PEGylation took place, asindicated by the absence of additional higher MW bands. Comparison tothe anti-Her2 Antibody (Anti-Her2-LysAzide274h) which shares an azide inthe same position of the heavy chain indicates the same Molecular weightshift for both bands and the same running time through the gel. Thenon-azide containing herceptin when treated with the 20KPEG alkyneshowed no reactivity. The conjugation efficiency for the anti-Her2Antibody (Anti-Her2-LysAzide274h70l) was also high, the gel showinglittle to no evidence of the unmodified heavy or light chains.

Example 10: PEGylation of her2 Antibodies Via Copper Catalyzed Click

PEGylation with 20KPEG Alkyne to Anti-Her2 Antibody(Anti-Her2-LysAzide274h) (See FIG. 60)

In a 200 uL PCR tube was placed a dichloromethane solution oftris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, 10 mM, 1.5 uL).The solvent was evaporated under a stream of nitrogen, and a solution of4D5 AzAb-2 (3.5 mg/mL, 2.14 uL) was added. An aqueous solution of 20KPEGalkyne was added (60 mg/mL, 1.67 uL), followed by an aqueous solution ofcysteine (20 mM, 0.5 uL). Finally, a solution of copper sulfate (10 mM,0.75 uL) was added and the mixture was vortexed gently to mixcomponents, then allowed to stand for 4h.

The solution was split (2.5 uL), with half being added to 7.5 uL ofreducing gel loading buffer, and half being added to 7.5 uL ofnon-reducing gel loading buffer. The samples were heated to 95 C for 3min and then loaded onto SDS-PAGE gels (4-20% Tris-Gly, Invitrogen) andrun. Non reducing gel (FIG. 11B) lane 2: anti-Her2 Antibody(Anti-Her2-LysAzide274h) untreated, Lane 3: anti-Her2 Antibody(Anti-Her2-LysAzide274h) treated with 20KPEG linear PEG alkyne. Amixture of unreacted anti-Her2 Antibody (NNAA lys-azide incorporated atposition 274 of heavy chain, a higher molecular weight band identifiedas the inclusion of one 20KPEG chain and a higher molecular weight bandidentified as the bis-PEGylated species were observed in thenon-reducing gel (lane 3). PDSI indicated a modest conversion betweenthe mono and bis PEGylated species. (5.3% bis PEGylation, 37%mono-PEGylation, 58% unmodified Ab). Reducing gel (FIG. 11A) Lane 2:antibody untreated, Lane 3: antibody treated with 20KPEG linear PEGalkyne. Gel analysis by PDSI indicated a modest amount (10%) of theheavy chain PEGylated. The reaction was specific for the heavy chain asthe light chain appears unaltered.

Additional examples of 20 kDa PEGylation of 4D5-AzAb(HC274) under CuAACconditions utilizing TBTA conditions are demonstrated in FIG. 32. Whencompared to the untreated AzAb, the PEGylation occurred in a sitespecific manner to the azide bearing heavy chain as indicated by asignificant molecular weight shift of this band.

PEGylation with 20K Linear PEG-Cyclooctyne to Anti-PSMA scFv with NNAASubstituted at Position 117 (Anti-PSMAscFV-117)

An scFv directed to PSMA was generated by grafting the CDRs of theantibody J591 (BANDER) onto a scFv framework. The scFv to PSMA wasgenerated by gene synthesis using overlapping oligomers and PCR and theproduct cloned into pJ201 to yield pJ201-PSMA. An expression constructpCDNA3.1-PSMA was generated by excising the ORF of the scFv byrestriction enzyme digest (XhoI and NotI) and the DNA fragment purified.The plasmid pCDNA3.1 was cut with the same enzymes and the PSMA scFvfragment inserted using T4 DNA ligase to produce pCDNA3.1-J591scFvcontaining a scFv to PSMA under control of the CMV promoter andcontaining an in frame 3′ 5×Pro-6×His tag (encoding PPPPPHHHHHH, SEQ ID81). To incorporate an amber codon into this scFv, site-directedmutagenesis was used to insert an amber stop codon following the last 3′codon of the scFv, but prior to the 5×Pro-6×His tag. The resultingconstructs named anti-PSMAscFV-117. Clones containing the amber codonwere identified by DNA sequencing.

Transient expression of the anti-PSMAscFv-117 containing a nnAA wereperformed in HEK293 cells stably expressing pylRS. This cell line wasgenerated by transfection of a vector containing the pylRS gene in pCEP4(Life Technologies) and selection by growth in medium containinghygromycin DMEM B (DMEM (DMEM (Life Technologies), 2 mM glutamax, 1 mMsodium pyruvate, 6 mM glutamine, 1× non essential amino acids (GibcoCAT#11140-050), 10% fetal calf serum, and 0.2 mg/mL hygromycin).Surviving cells were cloned by limiting dilution and clonesdemonstrating high functional activity of the pylRS were expanded. Thiswas achieved by transiently transfecting the different clones with avector encoding tRNApyl and a reporter construct GFPY40 containing anamber codon at position Y40 in the presence of ALOC nnAA. Fluorescencelevels were quantified in these cells using an Accuri flow cytometer andhigh functioning clones isolated. Expression of anti-PSMAscFv 117 wasperformed using standard transfection conditions. Cells were plated toapproximately 90% confluence and grown at 37 C. The following day, theplated cells were incubated with the appropriate DNA previously treatedwith a lipophilic reagent (Lipofectamine 2000, 293 fectin (invitrogen),according to the specific manufacturer's instructions. Following 2-5days of growth in the presence of 1-2 mM Lys-azide, the growth mediumwas harvested and either used directly or the expressed proteinspurified. Briefly, Expressed scFvs described here were purified fromgrowth medium following transient expression of eukaryotic cells. Ineach case 0.1 volumes of 10×PBS was added to the expression supernatantto equilibrate the salts and pH of the sample. The expressionsupernatant was dialysed to PBS at 4° C. for 16 h. Protein was bound toNickel-NTA beads (GE Healthcare) by batch binding or gravity flow andwashed extensively with wash buffer (50 mM sodium phosphate pH7.4, 300mM NaCl, 20 mM imidazole). Bound material was eluted with (50 mM sodiumphosphate pH7.4, 300 mM NaCl, 250-500 mM imidazole). Fractionscontaining the target protein were identified by SDS-PAGE and coomassiestaining. Peak fractions were pooled and dialysed against PBS prior tofurther use.

In a 200 uL PCR tube was placed a solution of anti-PSMA scFv with NNAAsubstituted at position 117 (0.3 mg/mL, 3.6 uL) was added followed by asolution of 20KPEG cyclooctyne (60 mg/mL, 1.0 uL). The solution wasmixed vigorously on a vortexer (Fisher). The tube was placed on a PCRtube centrifuge for a few seconds to place all liquids into the bottomof the tube. The mixture was allowed to stand for 4h.

The solution was diluted with reducing gel buffer (6 uL) to bring thefinal volume to ˜10 uL. The samples were then loaded onto SDS-PAGE gels(4-20% Tris-Gly, Invitrogen). SDS-PAGE (reducing) indicated that thePEGylation was successful consuming the majority of the anti-PSMA scFvband. Reducing gel (FIG. 12A) Lane 2: anti-PSMA scFv with NNAA lys-azideincorporated at position 117, untreated. Lane 3: anti-PSMA scFv withNNAA lys-azide incorporated at position treated with 20KPEG linear PEGcyclooctyne

To determine whether J591-scFv was functional and bound PSMA a cellbased fluorescence assay was used. Prostate cancer PC3 (PSMA negative)and LNCaP (PSMA positive) and the breast cancer cell line A345 weregrown in RPMI-1640. Cells were dissociated, counted and harvested bycentrifugation. For each assay 500,000 cells were incubated with PBScontaining 0.5% BSA for 1 hour at RT. Cells were then treated with 200uL of a pCDNA3.1-J591 transfection supernatant and washed with PBS. Thecells were then incubated with a Mouse anti-6×HIS antibody at 1 ug/mL(Clontech) for 1 hour at RT. Cells were washed with PBS and incubatedwith a Phycoerythrin conjugated anti-Mouse antibody (Miltenyi) for 30mins at RT. Cells were washed with PBS and analysed by flow cytometry(Accuri). Internalization assays were conducted above with the followingmodifications: Purified PSMA was utilized for internalization assays.During the incubation with the anti-PSMA scFv a cohort of cells was alsotreated with sodium azide (0.1%) at 4° C. for 1 hour to inhibit theinternalization of cell surface markers. Other incubations wereconducted at 37° C. In addition after the final wash, surface stainingwas inhibited by the addition of Trypan Blue to quench the phycoerythrinsignal.

Conjugation of Anti PSMA scFv with NNAA Substituted at Position 117(Anti-PSMAscFV-117) withMMAF-Valine-Citruline-p-Amino-Benzoyl-Carbonate-Cyclooctyne Derivative(See FIG. 61)

In a 200 uL PCR tube was placed a solution of phosphates (50 mM, pH=7.4,1 uL) and a solution of the anti-PSMAscFV-117 (40.5 uL, 2.1 mg/mL). Tothis was added a DMSO solution of theMMAF-valine-citruline-p-amino-benzoyl-carbonate-cyclooctyne derivative(3.46 uL, 13 mMol), the tube was capped and vortexed. The mixture wasallowed to stand for 4h. The reaction mixture was then treated with asolution of azidohomoalanine (AHA, 250 mM in 1M HEPES, 20 uL), vortexedand allowed to stand for 60 min. The mixture was then desalted through aZEBA (Pierce) mini spin column to afford the final scFv-drug conjugatesolution. Examination of the reaction mixture by SDS-PAGE (reducing)indicated a small molecular weight shift of the main PSMA bandconsistent with the conjugation of the drug moiety to protein. Theslight shift in PAGE gel is also observed with a separate scFvconstruct, 28D2 (FIG. 12).

Example 11: Testing of Decoy Amino Acids

Effective dnnAAs were identified by their ability to compete with a highaffinity substrate for pylRS (lys-azide) in an in vitro assay, for theirability to reduce background amber suppression levels observed with areporter protein, and for their ability to improve the viability andfunction of cells previously selected for high pylRS/tRNA activity.

To identify analogues that could effectively compete for pylRS bindingwith lys-azide, dnnAAs were first tested for function in an in vitrofunctional assay based on the ability of cells expressing WT pylRS andtRNApyl to introduce a nnAA at a target site in the reporter proteinGFPY40. This reporter contains an amber codon in its open reading framethat, in the absence of amber suppression, generates a truncated proteinthat is not fluorescent. In the event of amber suppression, a fulllength GFP protein is generated that is detectable by fluorescencedetection methods. In this assay, HEK293 cells stably expressing pylRS,were transiently transfected with a tRNA expression construct and theGFPY40 reporter cassette. Cells were then incubated with 0.5 mM lysazide in the presence or absence of different concentrations of dnnAAranging from 0.5 mM to 2 mM. GFP fluorescence levels were thenquantified by flow cytometry. For this assay the proportion offluorescent cells in each condition was determined and plotted. As thednnAAs are expected to compete with the nnAA (lys-azide) for pylRS/tRNAbinding, an effective dnnAA should reduce or prevent the expression offull length GFP (as described above dnnAAs can be delivered by the tRNAto the amber site, but cannot propagate protein synthesis). SeveraldnnAAs of Formula VII were tested: with the dnnAAs Formula VIIB.4 (FIG.13) increasing concentration of the dnnAA led to a concomitant reductionin the number of fluorescent cells suggesting a dose-related effect(FIGS. 13 A and B). To determine whether the dnnAA enabled amber codonreadthrough, transfected cells were also incubated with the dnnAA aloneand GFP expression monitored by flow cytometry dnnAA of Formula VIIB.4did not induce GFP expression. To control for non-specific effects ofthe dnnAAs, cells transfected with a reporter protein lacking an ambercodon, thus producing wildtype GFP (GFPwt) independent of ambersuppression, were also monitored in the presence of dnnAA (FIG. 13 A).dnnAA of Formula VIIB.4 (FIG. 13 A), showed no effect on proteinexpression, suggesting that the inhibition of amber suppression by thislatter dnnAA is specific.

A similar in vitro assay was used to gauge the effectiveness of thednnAAs of Formula VIIB listed in Table 1 where a description of theamino acids tested and a summary of the results are shown For this assaythe geometric mean fluorescence intensity of each sample was determinedby flow cytometry and plotted. For each dnnAA tested control cellstransfected with wild type GFP were measured as a positive control. Inaddition cells transfected with the reporter GFPY40 and exposed to 0.5mM lys azide or 2 mM lys azide were used to determine maximal GFPexpression levels in the absence of an inhibitor. In each case robustGFP expression levels were observed. To test whether any of the dnnAAcould reduce the efficacy of GFP expression, cells were incubated in thepresence of 0.5 mM, 1 mM and 2 mM lys azide. A reduction of GFPexpression concomitant with increased dnnAA concentrations was observedin all cases (FIG. 14). dnnAAs of Formula VIIB.1, Formula VIIB.3 andFormula VIIB.6 showed the greatest reduction of GFP expression relativeto samples lacking dnnAA (FIG. 14A, E, I). Their inhibition of GFPexpression increased with increasing concentrations of the dnnAAsuggesting that these are specific high affinity substrates for pylRS.dnnAAs of Formula VIIB.2 and Formula VIIB.5 also showed a reduction inGFP expression with increasing concentrations of dnnAA (FIG. 14C,G).However, the relative reduction in GFP expression was much lowersuggesting that these amino acid analogues have low affinity for thepylRS. Thus, dnnAAs of the present invention can compete with thelys-azide nnAA for binding to the pylRS and interfere with thephysiological mechanism of nnAA introduction. Cells transfected with thepylRS/tRNA pair and the GFPY40 reporter construct but not exposed toeither lys-azide or a dnnAA were used to determine levels of backgroundamber suppression and used as a negative control. In each case low GFPexpression levels were observed.

The competitive assay for dnnAA inhibition in the presence of a highaffinity substrate identified dnnAAs that compete for binding with tothe pylRS and thus are specific inhibitors of the pylRS/tRNA. However,the decoy nnAA is intended to reduce the levels of background ambersuppression in the absence of nnAA. To determine if background ambersuppression levels could be reduced, we incubated cells containing theGFPY40 reporter construct and the pylRS/tRNApyl pair in mediumcontaining one of the dnnAAs. To do this, HEK293 cells stably expressingpylRS were transiently transfected with a tRNA expression construct andthe GFPY40 reporter cassette. After 3 days of incubation, cells wereassayed for expression of GFP by flow cytometry and geometric meanfluorescence intensity of the samples determined. We have previouslyobserved that cells containing the full complement of the pylRS/tRNAamber suppression system show detectable expression of the GFP reporterconstruct that is above what is observed in cells lacking the ambersuppression system. This observation suggests that there isnon-orthogonal activity derived from the pylRS/tRNA pair that leads tohigher amber suppression levels than in cells lacking the pylRS/tRNApair. To identify dnnAAs capable of reducing background ambersuppression levels the transfected cells were incubated with each of thednnAAs at 0.5 mM, 1 mM and 2 mM and GFP levels measured by flowcytometry. The dnnAAs of Formula VIIB.1, Formula VIIB.3, Formula VIIB.6,Formula VIIB.2, and Formula VIIB.5 all showed reduction in thebackground amber suppression levels (FIG. 14D, F, J, D) relative tocontrol samples (cells not containing dnnAA (FIG. 14A-J;GFPY40+tRNA-nnAA)).

The decrease in background amber suppression dependent GFP expressionwas dose dependent and improved as the dnnAA concentration increased.Interestingly, one of the dnnAA, of Formula VIIB.2 showed very efficientinhibition of background amber suppression in this assay, reducing GFPfluorescence levels by 57.7% relative to control samples but had notbeen identified as a strong competitor of lys-azide. This suggests thatthe dnnAA of Formula VIIB.2 may have low affinity for the pylRS that iseasily displaced by lys-azide. This feature is an attractivecharacteristic for platform development as it enables the repression ofbackground amber suppression but the system can be activated uponaddition of a strong pylRS substrate such as lys-azide. These datasuggests that the dnnAA can occupy the pylRS-tRNA pair and prevent ambersuppression with natural amino acids. Formula VIIB.1 (62.5% resuction),Formula VIIB.3 (49.7%), Formula VIIB.6 (32%), and Formula VIIB.5 (35%)and Formula VIIB.4 (46.3%) were also effective in reducing backgroundamber suppression levels in this assay. Their efficacy was quantified bythe reduction in GFP expression relative to a control sample (notexposed to dnnAA) and the data are summarized in Table 1.

TABLE 1 Decoy nnAA of Formula VIIB % Reduction at 2 mM Structure IUPACname Formula Decoy

6-{[(prop-2-en-1- yloxy)carbonyl]amino}hexanoic acid VIIB.1 62.5

5-{[(prop-2-en-1- yloxy)carbonyl]amino}pentanoic acid VIIB.2 57.7

6-{[(2-chloroethoxy)carbonyl]amino}hexanoic acid VIIB.3 49.7

6-{[(tert-butoxy)carbonyl]amino}hexanoic acid VIIB.4 46.3

6-{[(prop-2-yn-1- yloxy)carbonyl]amino}hexanoic acid VIIB.5 35.0

6-{[(2-azidoethoxy)carbonyl]amino}hexanoic acid VIIB.6 32.7

Example 12. Effect of Decoy nnAA of the Invention on Platform Cell LineViability

To examine whether the dnnAAs of the invention could function to improvethe viability of cells containing pylRS and tRNApyl we monitored thegrowth and viability of a cell line, stably expressing pylRS andtRNApyl. For this assay CHO cells stably expressing pylRS and tRNApyland an IgG directed against her2/neu containing an amber codon in theheavy chain, shown to effectively incorporate nnAA into the expressedIgG thus producing an antibody containing a nnAA, were used for thisexperiment. Despite a high expression level of pylRS/tRNApyl pair, thiscell line has very robust cell growth characteristics when grown inmedium lacking nnAA. The presence of a highly expressed targetcontaining an amber codon likely has a protective effect on the cells bysupplying them with high levels of amber codons that absorb the ambersuppression activity and protects the cells from the effects ofbackground amber suppression at essential genes. However, upon additionof the nnAA (lys-azide) to the growth medium, and activation of theamber suppression machinery, a decrease in cell growth rate is observed.That is, the cell density of the culture appears to remain stable,suggesting that activation of the amber suppression machinery results ina cytostatic effect. To determine whether a dnnAA can rescue thiseffect, cells cultured in serum free medium were grown to a cell densityof 0.5×10⁶ cells/mL and subsequently treated with 0.5 mM lys azide aloneor in combination with 2 Mm dnnAA. Cell viability and cell numbers weremonitored daily over seven days. Cells treated with lys azide alonereached a cell density just below 1×10⁶ cells/mL on day 3 after nnAAaddition and remained at this density for the remainder of the assay(seven days) (FIG. 15A) The lack of growth was not likely due to loss ofviability as the culture retained high viability throughout theexperiment (˜70% viable cells) (FIG. 15B) Cultures treated with 2 mMCompound of formula VIIB.4, or 2 mM Formula VIIB.1 in combination with0.5 mM lys-azide supported continued growth of the culture that reachedover 1.5×10⁶ cells/mL and retained a cell viability of 90%. Cellstreated with the dnnAA of Formula VIIB.2 showed cell densities over3×10⁶ and viability well over 90% for the duration of the assay. Incontrast, cells treated with Formula VIIB.3 showed a decrease in cellviability over the course of the assay (<0.5×10⁶ cell/mL) and poorviability (30-40% by day 6). These data suggest that dnnAAs, of FormulaVIIB.2, Formula VIIB.4, and Formula VIIB.1 prevent the cytostaticeffects induced the activation of the amber suppression system. Cellsgrown in presence of the dnnAA of Formula VIIB.2 showed linear cellgrowth over time and reaching cell densities of 3×10⁶, over the sevendays. These data point the dnnAA of Formula VIIB.2 as the most efficientcompetitor of lys-azide for pylRS/tRNA function.

The data above showed that the dnnAA of Formula VIIB.2 is an efficientinhibitor of pylRS/tRNA and was shown to reduce the effects of ambersuppression dependent cytostasis in a cell containing a highly activeamber suppression machinery and expressing a target gene in the presenceof nnAA. However, the intended use of the dnnAA is in protecting cellswith highly active amber suppression machinery during their developmentand isolation. Thus, we next asked whether the dnnAA could improve theviability and performance of a cell pool enriched for a highly activeamber suppression machinery (platform cell line). To do this a platformcell line, selected for high activity of the amber suppression machinerywas grown in the presence or absence of dnnAA for several passages andsubsequently seeded into 96-well plates at ten cells per well and grownin the presence or absence of dnnAA (Formula VIIB.2). Each plate wasincubated for several days and cells harvested, pooled and counted.Interestingly, plates incubated with decoy nnAA showed higher cellnumbers than those grown in the absence of dnnAA (1.66×10⁶, and 1.5×10⁶cells/mL without dnnAA and 3.0 and 3.7×10⁶ cells/mL from cultures grownin dnnAA). This two fold increase in cell numbers may be due to theprotective effects of the dnnAA. To examine the activity of the cellsgrown under these conditions, 0.5×10⁶ cells pooled from each plate wereseeded into a 6-well plate and transfected with a GFPY40 reporterconstruct in the absence of dnnAA and with lys-azide. After 24 hours thefluorescence intensity of the cells in each sample was analysed by flowcytometry. The data were gated to include single cells and plotted todisplay the intensity of each event (scatter plot, FIG. 16). The numberof events falling within the top 10% of the GFP intensity spectrum weredetermined for each sample (Table 2). Cells grown in the presence of adnnAA showed higher numbers of events falling within the establishedgate (n=139 no decoy and n=175 with decoy (Formula VIIB.2). Thissuggests that more high activity cells were preserved by growth in decoynnAA containing medium. An additional metric was utilized to quantifythe performance of cells by isolating the geometric mean for the top 300events (Top 300) with highest GFP expression. Under this metric dnnAAincubated cells show improvement of performance over cells grown in theabsence of dnnAA (GM=954 without decoy; GM=1142 with decoy (FormulaVIIB.2). Cells from both groups were also transfected with a constructencoding wild type GFP. The same analyses were performed on this group.These data are summarized in Table 2 and indicate that cells grown indnnAA (Formula VIIB.2) containing medium show higher numbers of highlyfluorescent cells and higher fluorescence levels relative to the samecell line grown in the absence of dnnAA.

TABLE 2 dnnAA (Formula VIIB.2) increases amber suppression activity inplatform cell population: Sample Top 300 (GM) # events in top gate1-Tracer 343 25 6-Tracer 529 59 1 No decoy 1024 153 3 No decoy 885 125 5Decoy 1149 179 6 Decoy 1135 171

The dnnAA appeared to preserve the viability of the platform cells, butalso preserved cells with higher levels of amber suppressionfunctionality. To further assess the effect of dnnAA on cell growthcharacteristics of a platform cell line containing a highly active ambersuppression system we conducted a kinetic growth assay. To do this, theplatform cell line was incubated in the presence or absence of dnnAA in96-well plates as described above. Cells from each plate were pooled andseeded at 1000 cells per well in a 96 well plate in triplicate and cellsincubated for four days. On the fourth day Alamar Blue dye was added tothe cells and viability assayed by fluorescence emission. Alamar Blueserves as a convenient indicator of cell viability. Viable cellsmetabolize Alamar Blue producing resofurin which is a highly fluorescentdye. Fluorescence levels were monitored on days 5, 6, 9, and 11 and thefluorescence values plotted (FIG. 17). Decoy grown cells showed improvedcell viability compared to cells grown in the absence of dnnAA. This wasshown by fitting a line over the plotted growth rate and the slopes foreach calculated. Cells grown in decoy nnAA containing medium showedfaster growth rates (Avg slope=1190) than cells grown in the absence(Avg slope=669) of dnnAA. These data show that the use of a dnnAAprotects the cells from the chronic effects of amber suppression andimproves cell viability and growth of the culture. Taken together, thesedata point to dnnAAs as essential components for the development ofplatform cell lines and the preservation of cells with high ambersuppression activity.

Example 13: Translational Testing of Novel Pyrrolysine Analogs as nnAAswith a GFP Assay

An in vitro cell based assay was developed to assess the compatibilityof the pylRS/tRNA pair and the pyrrolysine analogs of the presentinvention (nnAAs) by and the efficiency of nnAAs integration into atarget protein. For this, HEK293 cells stably expressing pylRS (3H7)were transiently transfected with plasmids for the expression of tRNApyland a reporter construct encoding GFPY40 (containing amber codon inplace of tyrosine at amino acid residue number 40 (where 1 is theinitiator methionine)) using standard transfection protocols.Transfected cells were incubated with nnAAs at 2 mM for 2-3 days GFPproduction was analyzed qualitatively by visual inspection under themicroscope. The GFP fluorescence was quantified by flow cytometry usingan Accuri flow cytometer and the geometric mean of the fluorescent cellsdetermined.

This cell based assay was used to determine whether the different nnAAswere suitable substrates for the pylRS and allowed its translation intoa target protein. Cells expressing the PylRS/tRNApyl pair and containinga vector encoding the GFPY40 reporter gene were incubated in thepresence of the nnAAs. nnAAs that are readily utilized by thePylRS/tRNApyl pair support the translation of the nnAA into the ambersite of GFP and allow read-through of the gene producing full length GFP(fluorescent protein). The fluorescence intensity of the cells dependson the efficiency of nnAA incorporation. Thus, nnAAs that are poorlyutilized produce weakly fluorescent or non-fluorescing cells.Microscopic observation identified a number of nnAAs usable by the pylRS(Table 1, Positive GFP). Furthermore, the relative expression levels ineach sample was compared to those generated by substrates known to beefficiently utilized by pylRS. Formula V.1 (MFI=931,289), Formula V.2(MFI=1,676,250) and Formula V.3 (MFI=2,250,000) (see Table 3) supportedhigh levels of GFP expression with a geometric mean.

Analog Formulae VI.1 and VI.3 and of the present invention were found bythe inventors to be incorporated in the GFP reporter gene and yieldgreen cells under the experimental conditions used. Among these, theanalog of Formula VI.1 supported high levels of GFP expression (MFI904206) and represents an analogue that is efficiently utilized by thepylRS/tRNA pair under the experimental conditions tested (see Table 4).

TABLE 3 Formula V analog GFP results Formula IUPAC Name Positive GFP MFIV.1 (2S)-2-amino-6-{[(2- Yes 931289 azidoethoxy)carbonyl]amino}-hexanoic acid V.2 (2S)-2-amino-6-{[(prop-2-yn-1- Yes 1676250yloxy)carbonyl]amino}hexanoic acid V.3 (2S)-2-amino-6-{[(prop-2-en-1-Yes 2250000 yloxy)carbonyl]amino}hexanoic acid

TABLE 4 Formula VI analog GFP results Positive GFP Formula IUPAC NameAssay MFI VI.1 (2S)-2-amino-6-{[(2- Yes 904206azidoethyl)carbamoyl]oxy}hexanoic acid VI.3(2S)-2-amino-6-{[(prop-2-en-1- Yes yl)carbamoyl]oxy}hexanoic acid

Construction and Expression of Anti-Her2 Antibody

A full length anti-Her2 antibody containing two non natural amino acids(one in each heavy chain) (4D5-2AZ ab) was expressed in mammalian cells.A nnAA, containing an azide moiety, was incorporated at the selectedsites and purified by affinity chromatography using either protein Aresin (GE Healthcare) or by IgSelect (GE Healthcare, 17096901). Thepurified material was then concentrated and subjected to a conjugationreaction.

An antibody directed to the extracellular domain of Her2/neu wasgenerated by cloning the variable regions of both the heavy and lightchains of the mouse antibody 4D5 into vectors containing genes encodinghuman IgG. The variable regions of 4D5 were generated by gene synthesisusing overlapping oligomers and cloned into the human IgG1 frameworksencoded by pFUSE-CHIg-hG1 (IgG1 heavy chain; gamma) and pFUSE-CHLIg-hK(light chain; kappa; Invivogen) to generate a mouse-human hybrid. Ambercodons were introduced into the heavy chain (gamma) at positions K274 bysite directed mutagenesis. Clones containing the amber codon wereidentified by DNA sequencing. To generate an integrating construct thepromoters and ORF for the heavy chain was amplified by PCR and cloned byrestriction enzyme digestion and ligation into pOptivec (LifeTechnologies). The light chain and a single copy of the tRNA were joinedby two step PCR method using overlapping oligomers and cloned intoavailable sites into the pOptivec plasmid containing the heavy chain.The construct was then transfected into a CHO cell line containing thepylRS/tRNA pair and stably transfected cell lines showing highexpression of the IgG selected. This represents a second example of acell line stably expressing a mAb containing a nnAA indicating that theprocess has wide applicability for the use in the expression of mAbs.This cell line was utilized to generate IgG containing the nnAAsdescribed above. The cells were grown to a density of 1-2×10⁶ cells/mLin Excel DHFR-medium (Sigma-Aldrich) and nnAA added to culture to afinal concentration of 1 mM. Cells were incubated for 5 days and IgGpurified from the growth medium. Supernatants were harvested andsubjected to centrifugation to collect suspended cells and other debris.The supernatant was then filtered through a 0.22 um filter to remove anyparticulate material prior to application to a chromatography column.The filtered supernatant was applied to a 1 mL-5 mL prepacked HiTrapprotein A Sepharose at 1-5 mL/min flow rate using an AKTA chromatographysystem. The bound material and resin were washed with PBS to removeloosely bound proteins and the bound material eluted with 100 mM glycine(pH 3.0) at a flow rate of 1 ml/min. Peak fractions containing thetarget protein were neutralized with 0.1 fraction volumes of 1M Tris-HCl(pH8.0). All constructs were dialyzed to PBS at 4° C. for 16 hours intothe final phosphate buffer. The antibody with Formula VI.1 as nnAAincorporated into both of its heavy chains at position 274 was called“4D5-2AzAb-HC274-(2S)-2-amino-6-{[(2-azidoethyl)carbamoyl]oxy}hexanoicacid”.

PEGylation of4D5-2AzAb-HC274-(2S)-2-amino-6-{[(2-azidoethyl)carbamoyl]oxy}hexanoicacid

In a 200 uL PCR tube was placed phosphate buffer (5 uL, 500 mM, pH=7.4).A solution of4D5-2AzAb-HC274-(2S)-2-amino-6-{[(2-azidoethyl)carbamoyl]oxy}hexanoicacid (Formula VI.1). (10 uL, 0.55 mg/mL) was added followed by asolution of 20KPEG cyclooctyne (3.3, 60 mg/mL). The solution was mixedvigorously on a vortexer. The mixture was allowed to stand overnight.The mixture was diluted to 200 uL and applied to Protein-A magneticbeads. The mixture was vortexed and allowed to rotate to mix the beadsfor 90 min. The beads were immobilized and the run through materialdisposed. The beads were washed with PBS (2×) and then suspended inreducing gel buffer. Vortexed and then heated to 95 C for 3 min. Thesuspension was loaded directly onto an SDS-PAGE gel. Commassie stainingof the SDS-PAGE gel indicated the selective PEGylation of the Heavychain (FIG. 18, Lane 3).

Conjugation of4D5-2AzAb-HC274-(2S)-2-amino-6-{[(2-azidoethyl)carbamoyl]oxy}hexanoicacid with Fluorescence dye by SPAAC

In a 200 uL PCR tube was placed phosphate buffer (65 uL, 50 mM, pH=7.4).A solution of4D5-2AzAb-HC274-(2S)-2-amino-6-{[(2-azidoethyl)carbamoyl]oxy}hexanoicacid (30 uL, 0.55 mg/mL) was added followed by a solution DMCO-Fluor 488cyclooctyne (5.4, 5 mM in DMSO, click chemistry tools). The solution wasmixed vigorously on a vortexer. The mixture was allowed to stand for24h. The mixture was analyzed by HIC chromatography (Tosoh TSKgel ButylNPR with a gradient of 1M Sodium sulfate to phosphate buffer) showingthe conjugation had occurred and resulted in a mixture of DAR1 and DAR 2species (FIG. 19).

Example 14: Additional nnAA Data

Alternative Preparation of(2S)-2-amino-6-[[(2-azidoethyl)carbamoyl]oxy]hexanoic acid, Formula VI.1

Step 1:

In a 4 mL vial with magnetic stirrer was placedBoc-N-6-hydroxynorleucine (50 mg, 1 eq) and DMF (1 mL). To this wasadded 2-chloroethyl isocyanate (17.3 mg, 1.0 eq) and pyridine (32.3 uL,2 eq). The vial was capped and allowed to stir for 5h. The solution wastransferred to a extraction funnel, diluted with ethylacetate and 100 mMcitric acid. The mixture shaken and the layers separated. The aqueouslayer was extracted with ethyl acetate two additional times. The organiclayers combined, washed with 5% lithium chloride, dried with sodiumsulfate, filtered and concentrated. The product was taken forward intothe next step directly. Analytical MS: m/z (ES+) calculated 352.1(M+H)+. found 352.1.

Step 2:

In a 4 mL vial with magnetic stirrer was placed the crude chloroderivative from above and DMSO (1 mL). Sodium azide (130 mg, 5 eq) andpyridine (32.3 uL, 2 eq) were added to the mixture and the vial wascapped. The mixture was stirred overnight at 60° C. The mixture wastransferred to an extraction funnel and diluted with 100 mM citric acidand ethyl acetate. The mixture was shaken and the layers separated. Theaqueous layer was extracted with ethyl acetate two additional times. Theorganic layers combined, washed with 5% lithium chloride, dried withsodium sulfate, filtered and concentrated. The product was carried on tothe next step. Analytical MS: m/z (ES+) calculated 359.2 (M+H)+. found360.2.

Final Step:

In a 20 mL vial was placed the crude Boc protected amino acid andacetonitrile (2 mL). To this was added a solution of hydrochloric acidin dioxane (4N, 2.5 mL). The solution was stirred for 2h and thenconcentrated under reduced pressure. The mixture was lyophilized to asemi solid and used in translational testing. Analytical MS: m/z (ES+)calculated 259.1 (M+H)+. found 260.2.

Preparation of (2S)-2-amino-6-{[(prop-2-en-1-yl)carbamoyl]amino}hexanoicacid, Formula VI.3

In a 4 mL vial with magnetic stirrer was placedBoc-N-6-hydroxynorleucine (50 mg, 1 eq) and DMF (1.5 mL). To this wasadded allyl isocyanate (18.0 uL, 1.0 eq) and pyridine (32.3 uL, 2 eq).The vial was capped and allowed to stir for 4h. The solution wastransferred to an extraction funnel, diluted with ethylacetate and 100mM citric acid. The mixture shaken and the layers separated. The aqueouslayer was extracted with ethyl acetate two additional times. The organiclayers were combined, washed with 5% lithium chloride, dried with sodiumsulfate, filtered and concentrated. The product was identified by massspectrometry and taken forward into the next step directly. AnalyticalMS: m/z (ES+) calculated 330.2 (M+H)+. found 331.3.

In a 20 mL vial was placed the crude hydroxyl leucine-allyl carbamatederivative in acetonitrile (2 mL). To this was added a solution ofhydrochloric acid in dioxane (4N, 2.5 mL). The solution was stirred for2h and then concentrated under reduced pressure. The mixture waslyophilized to a semi solid and used in translational testing. Theproduct was confirmed by mass spectrometry. Additional purificationcould be done with ion exchange chromatography (DOWEX-50). AnalyticalMS: m/z (ES+) calculated 230.1 (M+H)+. found 231.2.

Example 15: IgG First Stable Cell Line

A cell line expressing Herceptin, capable of introducing a NNAA atposition 274 was constructed. DG44 CHO cells were transfected with twovectors, one containing the expression cassette for the heavy chain inpOptivec, and one for the light chain in pcDNA3.1 (hygro+) of Herceptin,and containing an amber codon at position H274. Cells were selected inmedium containing hygromycin and subsequently selected for expression bygrowth in medium containing Methotrexate. High expressing clones of thetruncated IgG were isolated by cloning. The best expressing clone wastransfected with a vector encoding pylRS and 18 copies of the U6-tRNApyl(pMOAV-2 puro). Transfected cells were selected by virtue of antibioticresistance and cells showing the highest amber suppression activityidentified through ELISA assays quantifying their full length IgGexpression after exposing clones to nnAA (lys-Azide). A clone showingstable expression of IgG containing nnAA at 12 ug/mL was isolated. Thisdata illustrates a third example of the construction of a mAb expressingcell line capable of nnAA incorporation by the pylRS/tRNA pair. Inaddition, this approach differs from the methods utilized previously inthe order of introduction of the functional elements.

Example 16: IgG Positional Mutations for Introduction of nnAAs

Example 5, the introduction of a mutation at heavy chain position 274 inthe anti-IL6 and Anti Her2 antibodies and the successful conjugation ofthe modified antibodies to various molecules were described.

Here, new IgG positional mutants and generation of DAR2 and DAR4 ADCsare described, from introduction of the mutation onto the cDNA toCytotoxicity data of the ADCs.

4D5 anti Her2 antibody was constructed with amber stop codons placedindividually at positions H274 and H359 of the heavy chain and L70 andL81 of the light chain. The H274, H359 and L81 were expressed asindividual mutants and H274 was also expressed with either L70 or L81 asdouble mutant in HEK293 cells. These 4D5 mutants were co-expressed withPyl-tRNA in HEK cells stably expressing PylRS. The supernatants werepurified on protein A and mAbs were PEGylated and analyzed by PAGE (FIG.20). The data indicate that PEGylation occurs efficiently at eachposition, with conjugation to multiple positions simultaneouslyoccurring as exemplified by the DAR4 species present in the reactionmix. 4D5-AzAb (HC274) and 4D5-4AzAb(HC359) undergo a clear molecularweight shift as a result of site specific PEGylation in the SDS-PAGEgel. Likewise, 4D5-AzAb (LC81) also shows a similar increase inmolecular weight as observable on PAGE gel. The heavy chain remainsuntouched (though distorted by residual PEG moving through the gel). TheHC274 and LC81 mutant containing four azides (4D5-AzAb (HC274-LC81))also readily PEGylated and was detectable by SDS-PAGE gel. Both theheavy and light chains show significant molecular shifts, similar tothose of the antibodies containing two azides (FIG. 20).

PEGylation of Positional Mutants

PEGylation of 20K Linear PEG-Cyclooctyne to 4D5-AzAb (LC81)

In a 200 uL PCR tube was placed a solution of 4D5-2AzAh (LC81) (8 uL,0.106 mg/mL) was added followed by a solution of 20KPEG cyclooctyne (2.0uL, 60 mg/mL). The solution was mixed vigorously on a vortexer. The tubewas placed on a PCR tube centrifuge for a few seconds to place allliquids into the bottom of the tube. The mixture was allowed to standfor 24h and then analyzed by SDS-PAGE (FIG. 20). Modification of thelight chain was evident by a clear molecular weight shift consistentwith the incorporation of a 20 kDa MW PEG (Lane 7).

PEGylation of 20K Linear PEG-Cyclooctyne to 4D5-AzAb (HC359)

In a 200 uL PCR tube was placed a solution of 4D5-AzAb (HC359) (8 uL,0.145 mg/mL) was added followed by a solution of 20KPEG cyclooctyne (2.0uL, 60 mg/mL). The solution was mixed vigorously on a vortexer. The tubewas placed on a PCR tube centrifuge for a few seconds to place allliquids into the bottom of the tube. The mixture was allowed to standfor 24h and then analyzed by SDS-PAGE (FIG. 20) The Azide containingantibodies with the azide at position 359 showed a clear molecularweight shift, specific to the heavy chain, as a result of site specificPEGylation (Lane 5).

PEGylation of 20KPEGcyclooctyne to 4D5-AzAb (HC274:LC70)

In a 200 uL PCR tube was placed a solution of 4D5-AzAb(HC274:LC70) (2uL, 0.47 mg/mL). A solution of 20KPEG cyclooctyne (1.0 uL, 60 mg/mL) wasadded and the solution mixed vigorously on a vortexer. The tube wasplaced on a PCR tube centrifuge for a few seconds to place all liquidsinto the bottom of the tube. The mixture was allowed to stand for 24hand then analyzed by SDS-PAGE (FIG. 20). Both the heavy and light chainsexperience a clear molecular weight increase in the PAGE gel as a resultof having a single PEG attached site specifically by the conjugation(Lane 6).

Conjugation of Cytotoxic Agents to Positional Mutants

Conjugation of 4D5-AzAb (LC81) with AF-Cyclooctyne derivative. In a 200uL PCR tube was placed a solution of 4D5-AzAb (LC81) (150 uL, 0.106mg/mL) and a DMSO solution of AF-Cyclooctyne (20 uL, 0.5 mMol), the tubewas capped and vortexed and allowed to stand for 24h. The reactionmixture was then treated with a solution of azidohomoalanine (AHA, 250mM in 1M HEPES, 20 uL), vortexed and allowed to stand for 2h. Themixture was then desalted through two mini ZEBA (Pierce) spin column toafford the final ADC solution. The mixture was analyzed by SDS-PAGE(FIG. 22) and HIC chromatography (FIG. 21). The resulting conjugateappeared as a single species in the HIC chromatogram and was slightlymore hydrophobic than the HC274 variant as determined by retention time.SDS-PAGE (Non-reducing) indicated a slight increase in MW as a result ofconjugating the drug (FIG. 22).

Conjugation of 4D5-AzAb (HC359) with AF-Cyclooctyne Derivative

In a 200 uL PCR tube was placed a solution of 4D5-AzAb (HC359) (150 uL,0.145 mg/mL) and a DMSO solution of AF-Cyclooctyne (20 uL, 0.75 mMol),the tube was capped and vortexed and allowed to stand for 24h. Thereaction mixture was then treated with a solution of azidohomoalanine(AHA, 250 mM in 1M HEPES, 20 uL), vortexed and allowed to stand for 2h.The mixture was then desalted through two mini ZEBA (Pierce) spin columnto afford the final ADC solution. The mixture was analyzed by HICchromatography (FIG. 21). The resulting conjugate appeared as a singlespecies in the HIC chromatogram and was significantly more hydrophobicthan the HC274 variant as determined by retention time. SDS-PAGE alsoindicated the formation of a band which was higher in molecular weightthan the parent antibody for the HC359 variant.

Conjugation of 4D5-AzAb (HC274:LC81) with AF-Cyclooctyne Derivative

In a 200 uL PCR tube was placed a solution of 4D5-AzAb (HC274: LC81)(150 uL, 0.187 mg/mL) and a DMSO solution of AF-Cyclooctyne (20 uL, 1mMol), the tube was capped and vortexed and allowed to stand for 24h.The reaction mixture was then treated with a solution ofazidohomoalanine (AHA, 250 mM in 1M HEPES, 20 uL), vortexed and allowedto stand for 2h. The mixture was then desalted through two mini ZEBA(Pierce) spin column to afford the final ADC solution. The mixture wasanalyzed by SDS-PAGE (FIG. 22) and HIC chromatography (FIG. 21). Theresulting conjugate appeared as a predominantly single species in theHIC chromatogram and was significantly more hydrophobic than the DAR2HC274. An increase in molecular weight was also observed in the SDS-PAGEunder non reducing conditions.

Conjugation of 4D5-AzAb (HC274:LC70) with AF-Cyclooctyne Derivative

In a 200 uL PCR tube was placed a solution of 4D5-AzAb (HC274: LC74) (50uL, 0.47 mg/mL) and a DMSO solution of AF-Cyclooctyne (5 uL, 3 mMol),the tube was capped and vortexed and allowed to stand for 24h. Thereaction mixture was then treated with a solution of azidohomoalanine(AHA, 250 mM in 1M HEPES, 20 uL), vortexed and allowed to stand for 2h.The mixture was then desalted through two mini ZEBA (Pierce) spin columnto afford the final ADC solution. The mixture was analyzed by SDS-PAGE(FIG. 22). An increase in molecular weight was observed in the SDS-PAGEunder non reducing conditions.

In Vitro Cytotoxic Activity

The ADC's generated as described above were tested for cytotoxicactivity in SKOV3 and HCC1954 and PC3 tumor cell lines which arestandard target cells for testing the activity of anti Her2 antibodiesand ADC cell lines. SKOV3 and HCC1954 express high levels of Her2, whilePC3 expresses Her2 at low level: the cytotoxic activity was calculatedas the concentration of ADC to kill 50% of the tumor cells in vitro asdescribed in Table 5. Notably, Herceptin alone exerts no cytotoxiceffect on any of the cell lines tested.

TABLE 5 the EC50 (in nM) are shown which represent the concentration ofthe drug to kill 50% of the tumor cells in vitro: EC₅₀ nM PC3 HCC1954SKOV3 HC-274 DNC 0.02123 0.1869 HC-274/LC-70 DNC 0.03059 0.1083HC-274/LC-81 DNC 0.01493 0.05233 HC-359 1.327 0.02414 0.1604 LC-81 1.1330.04365 0.201 AF 103.5 18.81 69.39 Herceptin DNC 0 0

As shown in FIG. 23 D, E, F, for each positional mutant, DAR2 and DAR4ADCs were compared. In each Her2 positive tumor cell line, the DAR4 ADCwas more potent than either DAR2, confirming the delivery of more drugwith the DAR4 than the DAR2 ADC.

FIG. 23 shows the cytotoxicity assay from which the EC50 values in Table5 were derived.

FIG. 23A shows the tumor cytotoxic activity of the 4D5-AzAb (HC274)-AFand 4D5-AzAb (HC359)-AF DAR2 ADC's as well as the 4D5-AzAb(HC274,LC-70)-AF DAR4 ADC in the SKOV3 tumor cell line. These cells areresistant to Herceptin alone but efficiently killed by the ADC withtoxin conjugated at different positions. Clearly, the ADC greatly lowersthat concentration of AF required to kill the tumor cells, presumably byefficiently targeting all of the AF directly to the cell, as compared topassive diffusion.

FIG. 23B shows the tumor cytotoxic activity of the 4D5-AzAb (HC274)-AFand 4D5-AzAb (HC359)-AF DAR2 ADC's as well as of the 4D5-AzAb(HC274,LC-70)-AF in HCC1954 cells. Similarly to SKOV3 cells, HCC1954cells are resistant to Herceptin, but efficiently killed by the ADC,with toxin conjugated at different positions.

FIG. 23C shows the tumor cytotoxic activity of 4D5-AzAb (HC274)-AF and4D5-AzAb (HC359)-AF DAR2 ADC's as well as the 4D5-AzAb (HC274,LC-70)-AFDAR4 ADC in the PC3 tumor cell line which expresses very low levels ofHer2 and is much more resistant to tumor killing by the ADC, as seen inthis figure as well as Table 5.

FIG. 23D shows the tumor cytotoxic activity in HCC1954 cells, a Her2overexpressing tumor cell line, of the 4D5-AzAb (HC274)-AF and 4D5-AzAb(LC-81)-AF DAR2 ADC's as well as the 4D5-AzAb (HC274,LC-81)-AF DAR4 ADC.As seen in the figure as well as Table 5, the DAR4 ADC is more potentthat either DAR2 constituents.

FIG. 23E shows the tumor cytotoxic activity in SKOV3 cells, a Her2overexpressing tumor cell line, of the 4D5-AzAb (HC274)-AF and 4D5-AzAb(LC-81)-AF DAR2 ADC's as well as the 4D5-AzAb (HC274,LC-81)-AF. As seenin the figure as well as Table 5, the DAR4 ADC is more potent thateither DAR2 constituents.

FIG. 23F shows the tumor cytotoxic activity in PC3 cells, a tumor cellline that expresses very low Her2, of the 4D5-AzAb (HC274)-AF and4D5-AzAb (LC-81)-AF DAR2 ADC's as well as the 4D5-AzAb (HC274,LC-81)-AF.As seen in the figure as well as Table 5, there is very little or noactivity of these ADC against this target.

Example 17: Pharmacokinetics, Stability, and In Vivo Anti Tumor Activityof Antibodies Conjugated at HC274 Position

Conjugation of Anti-Her2 Antibody (4D5 AzAb (HC274)) with DBCO-Fluor 488

In a 1000 uL HPLC vial equipped with magnetic stirrer was placed asolution of phosphates (511 uL, 50 mM, pH=7.4) and a solution of the4D5-AzAb (164 uL, 6.87 mg/mL). To this was added a DMSO solutionDBCO-Fluor 488 (75 uL, 10 mM in DMSO) the tube was capped and stirredfor 24h The reaction mixture was then treated with a solution ofazidohomoalanine (AHA, 250 mM in 1M HEPES, 50 uL), and stirred for 2h.The mixture was then desalted through a ZEBA (Pierce) 2 mL spin columnto afford the final antibody-dye conjugate. The material was assessed byHIC chromatography and SDS-PAGE. HIC chromatography indicated theformation of a single major species, consistent with the addition of twodye molecules per antibody (FIG. 24).

Rats were injected IV with 4D5 AzAb (HC274)-DBCO-Fluor 488 or Herceptinat a dose of 1 mg/kg, and serum levels of the two molecules weremonitored for 11 days using an anti-IgG ELISA.

As shown in FIG. 25A, the modification of IgG at the constant heavychain position 274 does not affect the pharmacokinetic profile asmeasured by serum levels and when compared to unmodified Herceptin.

The rat neonatal Fc receptor for IgG recognizes the human IgG Fc domainat the same residues as the human FcRn. Modified human IgG such as theADC's of the present invention, will be retained in vivo for extendedperiods of time, due to the function of the rat FcRn, as long as theinteraction of the conjugate and the FcRn remains intact. These datademonstrate that HC-274 modified IgG retains a long in vivo half life inrat, indicating that the FcRn Interaction is not blocked by theconjugate. The same residues on 4D5 that interact with the rat FcRn arealso responsible for the interaction with human FcRn. These data showthat the ADC with a conjugate at HC-274 will interact with the humanFcRn and therefore retain a long half life in man.

The same sera collected for the PK analysis shown in FIG. 25A weretested for the presence of the FITC conjugate on the 4D5 IgG (FIG. 25B)by a quantitative ELISA assay in which the antibody is captured by Her2extracellular domain protein coated on plastic ELISA wells. Afterincubation, the wells are washed and incubated with anti-FITC antibodyconjugated to HRP. After incubation, the wells are washed and HRPsubstrate added. This ELISA measures the amount of FITC remaining on theADC, and is reported as ng/ml of ADC with all the FITC intact, as shownin FIG. 25B. The 4D5 ADC in the sera with DAR2 is the same level as theuntreated ADC, indicating no loss of FITC. The data clearly indicatethat the dye is completely retained and stable in vivo in rat for thefull 11 days of the study.

Conjugation of 4D5-AzAb (HC274) with AF-Cycloalkyne Derivative.

In a 1000 mL HPLC tube with magnetic stirrer was placed a solution ofphosphates (24 uL, 50 mM, pH=7.4) and a solution of the 4D5-AzAb (149uL, 6.87 mg/mL). To this was added a DMSO solution of 5 (27.2 uL, 2mMol), the tube was capped and vortexed. The mixture was allowed tostand for 24h. The reaction mixture was then treated with a solution ofazidohomoalanine (AHA, 250 mM in 1M HEPES, 50 uL), vortexed and allowedto stand for 60 min. The mixture was then desalted through a ZEBA(Pierce) 2 mL spin column to afford the final ADC solution. The mixturewas analyzed by HIC chromatography and SDS-PAGE (FIGS. 26A and B). HICchromatography indicated the formation of a major species consistentwith two auristatin molecules being added per antibody. SDS-PAGE(reducing) indicated a small molecular weight shift to the heavy chainconsistent with the addition of a auristatin molecule being added.

In Vitro Activity

4D5-AzAb (HC274)-AF was tested for its in vitro potency on Her2 positivecell lines. The Her2 over expressing cell lines, SKBR3 and SKOV3 werecompared to PC3, a cell line that expresses very low level of Her2.4D5-AzAb (HC274)-AF was compared to auristatin (AF) alone. The ADCspecifically killed the SKBR3 and SKOV3 cells that overexpress Her2 butdid not kill PC3 cells which expresses very little Her2. The potency ofthis ADC against the 3 target cell lines is shown in Table 6. Thesevalues were calculated on the cytotoxicity data shown in FIG. 27. Whilethe ADC shows picomolar potency against SKOV3 and SKBR3, it is inactiveagainst PC3, even though that cell line is quite sensitive to auristatinalone. This demonstrates the specificity of these ADC for cells thatexpress high levels of Her2.

TABLE 6 Potency of 4D5 auristatin conjugate for Her2 positive tumor celllines in vitro: EC₅₀ nM PC3 SKOV3 SKBR3 4D5-AF — 0.019 0.0074 Auristatin336 287 71

In Vivo Antitumor Activity

SKOV3 tumor cell line is derived from human ovarian carcinomaoverexpressing Her2 but resistant to Herceptin; tumors derived fromSKOV3 cells were established in acid mice. The tumors reachedapproximately 100 mm³ within 2 weeks, and at that time, the mice wererandomized and half the mice (n=4/group) were treated with a singlesubcutaneous injection of 6 mg/kg of the 4D5-2AzAb (HC274)-AF ADC (FIG.28). Tumor progression was followed by caliper measurement of the tumorsize. All the treated mice showed highly significant delay in tumorprogression after a short period of tumor shrinkage. One mouse wascompletely cured while the other three mice eventually relapsed (FIG.28B). Tumor progression was monitored for up to 80 days to ensure thatthe single cured mouse did not relapse.

This example demonstrates that the 4D5-AF ADC is retained in circulationin vivo, stable to metabolic degradation, but available to deliverpotent toxic activity specifically to the cytoplasm of the target tumorcells.

Example 18:—Generation of Bispecific Antibodies/Antibody Fragments andCharacterization

Preparation of 4D5 AzAb-0.5KPEG Intermediate. In a 200 uL PCR tube wasplaced phosphate buffer solution (34.3 uL, 50 mM, pH=7.4). To this wasadded a solution of 4D5 2-AzAb(HC274) (23.61 uL, 13 mg/mL) and asolution of bis-cyclooctyne linker (2.04 uL, 20 mM in dioxane, 500 kDa).The mixture was vortexed intermittently over a 24h period. The mixturewas purified by CHT resin to afford the functionalized intermediate.

28D2 scFv-AHA is derived from the anti-IL6 antibody in Example 4. A fullSEQ ID and description of preparation can be found in WO12032181.Briefly, the antibody fragment, 28D2 scFv-AHA is expressed in e. coliand the nnAA azidohomoalanine is incorporated at the C-Terminus of thesequence. The protein is isolated from the fermentation and purified bynickel affinity chromatography.

Preparation of 4D5 AzAb (HC274)-28D2 (scFv) Bispecific

In a 200 uL PCR tube was placed 4D5-0.5KPEG conjugate (37.5 uL, 2 mg/mL)and a solution of 28D2 scFv-AHA (22 uL, 4.6 mg/mL). The mixture wascapped, vortexed and allowed to stand for 24h. The mixture was purifiedby Protein A magnetic Beads (GE), analyzed by SDS-PAGE (FIG. 29).SDS-PAGE indicated the formation of two distinct bands higher inmolecular weight than the starting azide containing antibody which wouldbe consistent with the addition of one and two scFv molecules.

In the ELISA assay, an anti IgG antibody was affixed to a solid surface.The Her2-anti-IL6 bispecific was captured on the Fc region of thebispecific. The bispecific was then assessed for function by theaddition of IL-6 which was detected in turn by biotin labeling.

In one ELISA assay, the ability of the bispecific to bind the Her2antigen was probed. The bispecific was found to have a similar level ofantigen affinity to the control 4D5 AzAb (HC274) antibody (FIG. 30C).

In a second ELISA assay, the ability of the bispecific to bind to IL-6was probed. In this version of the ELISA, the bispecific was captured onthe ELISA plates by anti-IgG interaction. The IL-6 affinity was theninvestigated. It was found that the bispecific possessed a similar levelof affinity for IL-6 as a full length antibody, 13A8 (FIG. 30B).

In the final ELISA assay, the ability of the bispecific to function atboth ends at the same time was probed. The bispecific was captured ELISAplate by the antibody affinity for the Her2 antigen. The IL-6 activitywas then probed. It was found the bispecific possessed high affinity forthe Her2 antigen and IL-6 at the same time. The control antibodies wereunable to show similar bifunctional activity (FIG. 30A).

The FGF21 polypeptide (FGF21-AHA(s86)) was expressed in e. coli andmodified at position 86 of SEQ ID 62 to incorporate azidohomoalanine toreplace serine. The modified protein was isolated as inclusion bodiesand purified by nickel affinity chromatography.

Preparation of 4D5 AzAb-FGF21 (Cytokine) Bispecific

In a 200 uL PCR tube was a solution of the Antibody-Linker intermediate(3.0 uL, 2 mg/mL) and a solution of FGF21-AHA (S86) (28.6 uL, 2.8mg/mL). The tube was capped and incubated at 37 C for 24h. The mixturewas purified by Protein A magnetic beads and analyzed by SDS-PAGE (FIG.31). A molecular weight shift was observed in the SDS-PAGE gelconsistent with the addition of the FGF21 (S86) molecule to the heavychain by way of the intermediate linker.

Example 19. PEGylation of 4D5 Azab with Copper Promoted Azide AlkyneCycloaddition and the Tris(3-Hydroxypropyltriazolylmethyl)Amine (THPTA)Ligand

20 kDA PEGylation with THPTA. In a 200 uL PCR tube was placed a solutionof phosphate buffer (3 uL, 150 mM, pH=7.4). To this was added a solutionof 4D5 azide containing antibody (6.5 uL, 4.6 mg/mL) and a solution of20 kDa PEG alkyne (4 uL, 60 mg/mL). In a separate tube was placed asolution of copper sulfate (2.0 uL, 10 mM), and solutions of THPTA (2.5uL, 40 mM), amino guanidine (1.0 uL, 100 mM) and sodium ascorbate (1.0uL, 100 mM). The tube was capped, vortexed and allowed to stand for 10min. The entire copper complex was added to the AzAb-Alkyne solution.The final mixture was capped, vortexed and allowed to incubate for 2h at37° C. or 50° C. The reaction was analyzed by SDS-PAGE (FIG. 33).SDS-PAGE indicated a molecular weight shift of the heavy chainconsistent with the addition of a 20 kDa PEG.

2 kDA PEGylation with THPTA. In a 200 uL PCR tube was placed a solutionof 4D5 azide containing antibody (4.9 uL, 13 mg/mL) and a solution of 20kDa PEG alkyne (4.2 uL, 20 mM). In a separate tube was placed a solutionof copper sulfate (3.5 uL, 20 mM), and solutions of THPTA (8.8 uL, 40mM), amino guanidine (3.5 uL, 100 mM) and sodium ascorbate (3.5 uL, 100mM). The tube was capped, vortexed and allowed to stand for 10 min. Aportion of the copper complex (1.93 uL) was added to the AzAb-Alkynesolution. The final mixture was capped, vortexed and allowed to incubatefor 2h at 37° C. or 60° C. The reaction was analyzed by SDS-PAGE (FIG.34) and HIC chromatography. SDS-PAGE reducing showed a slight increasein molecular weight of the heavy chain consistent with the addition of a2 kDa molecular weight PEG. SDS-PAGE under non-reducing conditionsindicated a small molecular weight shift of the full length antibodyconsistent with the addition of PEG. Additional confirmation wasprovided by HIC chromatography (FIG. 34B), which indicated a singlemajor species, consistent with the addition of PEG to the antibody.

Example 20. Preparation of Additional Cytotoxin-Alkyne Derivatives

Preparation of Amanitin-Cyclooctyne Derivative

α-Amanitin (5 mg, 5.5 umol) and glutaric anhydride (1.5 mg, 13.2 umol)and pyridine (500 uL) were placed in a 2 mL vial with magnetic stirrer.The solution was stirred overnight. The mixture was concentrated tounder vacuum taken up in a small amount of dichloromethane andprecipitated in methyl tbutyl ether. The solids were carried forwardinto the next step. Analytical MS: m/z (ES+) calculated 1031.4 (M+H)+.found 1033.3.

Amanitin-GA (5.6 mg, 5.5 umol), HBTU (4.6 mg), cyclooctyne-amine (1.8mg) and triethylamine (1.9 uL) were placed in a 5 mL centrifuge tube anddissolved in DMF (1 mL). A small magnetic stir bar was added was addedand the mixture was stirred overnight. The mixture was precipitated fromMethyl tButyl ether. The solids were isolated by centrifugation.Analytical MS: m/z (ES+) calculated 1337.6 (M+H)+. found 1339.4.

Preparation of Auristatin F-Cyclooctyne Derivative

In a 4 mL vial with magnetic stirrer was placed Auristatin F (AF) (5 mg,10. umol) in DMF (1 mL). To this mixture was added HBTU (7.6 mg, 20umol), BCN amine (3.2 mg, 10 umol) and triethylamine (3.4 uL, 25 umol).The vial was capped and the mixture allowed was stirred overnight. Thesolution was purified by reversed phase HPLC (acetonitrile/water 0.1%TFA gradient). The desired fractions pooled and lyophilized to a powder.Analytical MS: m/z (ES+) calculated 1051.7 (M+H)+. found 1052.7.

Auristatin F-Propargylamide Derivative AF-PA0

AF-PA0 refers to the PEG spacer between the alkyne and auristatin F. ForAF-PA0 there is no PEG spacer, hence the zero. AF-PA3 incorporates threeethylene units between the alkyne and the auristatin F structure.

In a 4 mL vial was placed Auristatin F (AF) (6.1 mg, 8.18 umol) in DMF(1 mL). To this mixture was added a solution of HBTU (6.2 mg, 16 umol),propargylamine (0.6 uL, 9 umol) and triethylamine (2.8 uL, 20 umol). Thevial was capped and the mixture allowed to incubate overnight. Thesolution was purified by reversed phase HPLC (acetonitrile/water 0.1%TFA gradient). The desired fractions pooled and lyophilized to a powder.Analytical MS: m/z (ES+) calculated 782.5 (M+H)+. found 783.3.

Auristatin F-Propargylamide Derivative AF-PA3

In a 1 mL vial with magnetic stirrer was placed Auristatin F (AF) (4.7mg, 6.3 umol) in DMF (200 uL). To this mixture was added a solution ofHBTU

(6.0 mg, 16 umol in 50 uL DMF), prop-2-yn-1-ylN-{2-[2-(2-aminoethoxy)ethoxy]ethyl}) carbamate (3.7 mg, 14 umol in 100uL DMF) and triethylamine (3.7 uL, 25 umol). The vial was capped and themixture allowed to stir overnight. The solution was purified by reversedphase HPLC (acetonitrile/water 0.1% formic acid gradient). The desiredfractions pooled and lyophilized to a powder. Analytical MS: m/z (ES+)calculated 957.6 (M+H)+. found 958.5.

Example 21: Conjugation to Anti-Her2 Antibodies with Toxin-AlkyneDerivatives

Conjugation of 4D5-AzAb (HC274: LC70) with AF-Cyclooctyne Derivative.

In a 200 uL PCR tube was placed a solution of 4D5-AzAb (HC274: LC70) (24uL, 0.5 mg/mL) and a DMSO solution of AF-Cyclooctyne (0.8 uL, 10 mMol),the tube was capped and vortexed and allowed to stand for 24h. Thereaction mixture was then treated with a solution of azidohomoalanine(AHA, 250 mM in 1M HEPES, 20 uL), vortexed and allowed to stand for 2h.The mixture was then desalted through ZEBA (Pierce) spin column toafford the final ADC solution. The mixture was analyzed by SDS-PAGE.

4D5-AzAb (HC274: LC70)-AF and 4D5-AzAb (HC274)-AF were assessed by an invitro potency assay for their ability to kill Her2 positive cells. Thein vitro assay is described in example 16. Briefly, the ADC was comparedto the unconjugated antibody (herceptin) and the free drug. In thecytotoxicity assay, the DAR4 ADC was found to be slightly more potentthan the related DAR2 (4D5AzAb(HC274)-AF) compounds described in example15 and FIG. 23 versus Her2 positive expressing cell lines such as SKOV3and SKBR3. The compounds showed minimal activity versus a low Her2expressing cell line such as PC3. Both ADC's were more potent than theunconjugated antibodies or the free drug (FIG. 35, A,B,C) Conjugation ofanti-Her2 Antibody (4D5-AzAb (HC274)) with Amanitin-cyclooctynederivative

In a 200 uL PCR tube was placed a solution of phosphates (5 uL, 50 mM,pH=7.4) and a solution of the 4D5-AzAb(HC274) (3.94 uL, 13 mg/mL). Tothis was added a DMSO solution of amanitin alkyne (1.13 uL, 3 mMol), thetube was capped and vortexed. The mixture was allowed to stand for 24h.The reaction mixture was then treated with a solution ofazidohomoalanine (AHA, 250 mM in 1M HEPES, 7 uL), vortexed and allowedto stand for 2h. The mixture was then desalted through a ZEBA (Pierce)mini spin column to afford the final ADC solution 4D5-AzAb(HC274)-Amanitin.

The 4D5-AzAb (HC274)-Amanitin was assessed by an in vitro potency assayfor the ability to kill Her2 positive cells. The in vitro assay isdescribed in example 16. Briefly, the ADC was compared to another ADC(4D5AzAb(HC274)-AF) and the free drug. 4D5AzAb(HC274)-amanitin ADC wasactive in Her2 positive cell lines SKBR3 (FIG. 36A) whilst showedminimal activity in the low Her2 expressing cell line such as PC3 (FIG.36B). 4D5-AzAb (HC274)-Amanitin was similarly potent as4D5AzAb(HC274)-AF and more potent than the free drug alone.

Example 22: Conjugation to 4D5 2AzAb (HC274) to AF-Alkyne with CuAAC

Conjugation of 4D5-2AzAb (HC274) with AF-PA0 or AF-PA3. In 200 uL PCRtubes was placed phosphate buffer (3.0 uL, 50-500 mM, pH=7.4), asolution of 4D5-AzAb (HC274) (2.1 uL, 25 m/mL in PBS) and a organicsolution of the cytotoxic agent, AF-PA0 or AF-PA3 (0.70 uL, 5 mM, inDMSO or propylene glycol). In a separate tube was placed a solutions ofcopper sulfate (7 uL, 10-160 mM), THPTA (3.6 uL, 10-160 mM), aminoguanidine (7.0 uL, 10-200 mM), and sodium ascorbate (7.0 uL, 50-300 mM).The THPTA-CuSO4 complex was capped, vortexed and allowed to stand for 10min. The copper complex (1.23 uL per rxn) was added to the AzAb-Alkynesolutions. The final mixture was capped, vortexed and allowed toincubate (4 C to 60 C) for 0.5-18h. The material was desalted by passingthrough a Pierce Zeba mini spin column (Cat#89882, MWCO=7000) andtreated with 10×PBS. Alternatively, the reactions mixtures are purifiedby CHT chromatography.

Conjugation of 4D5-2AzAb with AF-PA0 at 1:1 THPTA: CuSO4 Ratio at roomtemperature. In 200 uL PCR tubes was placed phosphate buffer (2.8 uL,500 mM, pH=7.4), 4D5-AzAb(HC274) (2.1 uL, 25 mg/mL) and AF-PA0 (0.7 uL,5 mM, DMSO solution). In separate tubes were placed a solutions ofcopper sulfate (3.5 uL, 20 mM), THPTA (1.8 uL, 40 mM), amino guanidine(3.5 uL, 100 mM), and sodium ascorbate (5.3 uL, 100 mM). The THPTA-CuSO4complex was capped, vortexed and allowed to stand for 10 min. The coppercomplex (1.4 uL) was added to the AzAb-Alkyne solutions. The finalmixture was capped, vortexed and allowed to incubate for 1 h at roomtemperature. The reactions were purified by desalting through a ZebaSpin column (MWCO=7000). Analysis of the reaction by HIC chromatographyindicated clean formation of the desired DAR2 product. (FIG. 38).

The CuAAC based anti-Her2 auristatin ADC was compared to the relatedcycloalkyne derived anti-Her2 auristatin ADC by an in vitro assay tomeasure potency and selectivity for. The in vitro potency assay was runin a similar manner to that described in example 16. The CuAAC based ADCwas found to have a similar potency to the cycloalkyne derived ADCversus Her2 positive cell lines such as SKBR3 and SKOV3 (FIG. 37A, B).The same ADC's were tested for selectivity versus a low expressing Her2cell line, PC3 and found to be non potent (FIG. 37C).

TABLE 7 Summary of CuAAC condition utilized for AzAb conjugations: AzideCopper Rxn containing Alkyne Copper stabilizing Reducing Componentproteins substrate Source Ligand Agents Examples PSMA-azide CytoxicCuSO4 THPTA BME scFv-azide agents CuI TBTA Cysteine α-IL6 AzAb Dye CuCl2MES TCEP α-Her2 AzAb PEG (2- Cu(Ac)2 ET3N Sodium 20 kDa) CuBr ascorbateProtein-PEG Sodium bisulfite Conjugates Hydrazine hydroxylamine ConcRange 0.001-0.1 0.001-5 0.1-10 0.2-20 0.1-30 (mM)

Example 23 Generation of Herceptin ADC and Conjugation

The cell line described in Example 15 was used to generate an anti-Her2azAb antibody derived from Herceptin (SEQ ID 74 and 75, light chain; SEQID 76, 77, heavy chain) modified to contain a nnAA at position 274 ofthe heavy chain (mutant heavy chain, SEQ ID 76,77; unmodified lightchain as per SEQ ID 78,79), Herceptin AzAb(HC274). 3×10⁶ cells/mL wereseeded into 125 mL of Excell DHFR-medium and exposed to lys-azide for 7days. The medium was collected and cells harvested by centrifugation(1000×g for 10 min). 12.5 mL of 10× phosphate buffered saline (10×PBS;Life Technologies) was added and medium passed three times over a 300 ul(packed volume) IgSelect resin (GE Healthcare). The bound protein waswashed with 10 column volumes of Tris buffered saline containing 0.1%tween-20 (TBS-T pH7.5). Herceptin-AzAb was eluted with 0.1M glycinepH2.5 and 250 ul elution fractions collected. The acid was immediatelyneutralized with 50 ul 1M Tris pH8.0. Each fraction was analysed byspectrophotometry and fractions showing OD280 readings were retained.Peak protein fractions were combined and protein concentrated andbuffered exchanged into phosphate buffered saline using an AmiconUltra-4 concentrator (Millipore). Concentrated samples were processedfor conjugation.

PEGylation of 20K Linear PEG-cycloalkyne to Herceptin-AzAb (HC274). In a200 uL PCR tube was placed a solution of Herceptin-2AzAb (HC274) (1.0uL, 2.4 mg/mL) followed by a solution of 20KPEG cyclooctyne (1.0 uL, 60mg/mL). The solution was mixed vigorously on a vortexer. The tube wasplaced on a PCR tube centrifuge for a few seconds to place all liquidsinto the bottom of the tube. The mixture was allowed to stand for 4h andthen analyzed by SDS-PAGE. SDS-PAGE (reducing) indicated the 20 kDa PEGalkyne was site specifically conjugated to the azide of the heavy chainwith excellent efficiency, with minimal to no unreacted heavy chainremaining (FIG. 39).

Conjugation of Herceptin-AzAb(HC274) with AF-Cyclooctyne derivative. Ina 200 uL PCR tube was placed a solution of the Herceptin-2AzAb (19 uL,4.8 mg/mL). To this was added a DMSO solution of AF-cyclooctynederivative (1.5 uL, 5 mMol), the tube was capped and vortexed. Themixture was allowed to stand for 24h. The reaction mixture was thentreated with a solution of azidohomoalanine (AHA, 250 mM in 1M HEPES, 10uL), vortexed and allowed to stand for 60 min. The mixture was thendesalted through a ZEBA (Pierce) 2 mL spin column to afford the finalADC solution. Analysis by HIC chromatography indicated the cleanformation of the desired DAR2 product (FIG. 40). Additional analysis bySDS-PAGE (reducing) indicated a small increase in molecular weight ofthe heavy chain, non reducing PAGE also indicated an increase inmolecular weight of the main protein band.

Conjugation of Herceptin-2AzAb with AF-PA0 under CuAAC conditions. Theconjugation was done under conditions described in example 22. Thereactions were purified by desalting through a Zeba Spin column(MWCO=7000). Analysis of the reaction by HIC chromatography indicatedclean formation of the desired DAR2 product (FIG. 41). Additionalanalysis by SDS-PAGE (reducing) indicated a small increase in molecularweight due to conjugation of the drug to this subunit. Additional PAGE(non-reducing) analysis also indicated a molecular weight increase ofthe main protein band (FIG. 41).

In Vitro Cytotoxic Activity

The ADC's generated as described above were tested for cytotoxicactivity in SKOV3 and a PC3 tumor cell lines which are standard targetcells for testing the activity of anti Her2 antibodies and ADC celllines. SKOV3 cells express high levels of Her2, while PC3 cells expressHer2 at low level. Briefly, for each assay 1000 cells are seeded intoeach well of a 96 well plate and incubated with a titration ofAuristatin F alone, or Herceptin-AF conjugates generated by either SPAACor CUAAC chemistry. The drug treated cells are incubated at 37 C for 3days in 100 ul medium. 20 ul of Alamar Blue (Life Technologies) is addedto each well and the cells incubated for 16-24 hours and an OD 450 nmdetermined for each well. The cytotoxic activity of the conjugates wascalculated as the concentration of ADC to kill 50% of the tumor cells invitro as described in Table 8.

TABLE 8 Potency of Herceptin ADC against different tumor cell lines EC₅₀nM PC3 SKOV3 HCC1954 BT474 SKBR3 Herceptin-CUAAC-AF NA 0.030 0.023 0.0690.017 ADC Herceptin-SPAAC-AF NA 0.026 0.023 0.062 0.017 ADC Auristatin F166.2 23.48 12.9 58.81 33.27

As shown in FIGS. 42 A and B, Herceptin-AzAb (HC274)-AF ADCs constructedwith CUAAC or SPAAC conjugation chemistries were compared. In each casethe Herceptin ADC was potent in SKOV3 cells (Her2 positive tumor cellline), but did not affect PC3 cells.

FIGS. 42A and B shows the cytotoxicity assay from which the EC50 valuesin Table 8 were derived.

FIG. 42A shows the tumor cytotoxic activity of the Herceptin-AzAb(HC274)-CUUAC-AF and Herceptin-AzAb SPAAC-AF in the SKOV3 tumor cellline. These cells are efficiently killed by the ADC with toxinconjugated generated by the different conjugation chemistries. Clearly,the ADC greatly lowers that concentration of Auristatin F required tokill the tumor cells, presumably by efficiently targeting all of the AFdirectly to the cell, as compared to passive diffusion. FIG. 42B showsthe effect of the Herceptin conjugates on PC3 cells. Here, both SPAACand CUUAC generated conjugates did not show cytotoxicity at the examinedconcentrations. These data show specific targeting and activity of theHerceptin ADCs generated by both CUUAC and SPPAAC conjugation methods.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

All patents and patent applications referred to herein are incorporatedby reference in their entirety.

REFERENCES

-   1. Blight et aL. 2004 Nature. 431 333-335 (2004)-   2. Chen, P., 2009 Agnew Chem Int Ed Engl. 48, 4052-55.-   3. Hancock et AL. JACS 2010, 132, 14819-24-   4. Hecht et al., 1978 JBC 253, 4517-20.-   5. Herold et al., 2008 PNAS 105, 18507-12.-   6. Kavran et al., 2007 PNAS 104, 11268-73.-   7. Kohrer et al., 2001 PNAS 98, 14310-15;-   8. Kohrer et al., Chem & Biol., (2003) 10, 1095-1102;-   9. Liebman S W. and Sherman, F. 1976 Genetics, 82, 233-249.-   10. Liebman, S W et al., 1976 Genetics 82, 251-272.-   11. Liu W. et al. 2007, Nature methods, 4; 239-244.-   12. Mukai et al 2008 BBRC 371, 818-823-   13. Naykova et al. 2003 J Mol. Evol. 57:520-532.-   14. Neumann et al. 2008 Nat. Chem. Biol. 4, 232-234.-   15. Nguyen et al., 2009 J. Am. Chem. Soc. 131 (25), pp 8720-8721-   16., Nozawa 2009, Nature. 457 1163-67.-   17. Pettit et al., 1997 Fortschr. Chem. Org. Naturst 70, 1-79-   18. Sakamoto, K. 2002 Nucl. Acid Res. 30, 4692-4699.-   19. Shan L. et al., J Gene Med. (2006) 8, 1400-1406.-   20. Senter P. et al., 2003 Blood 102, 1458-65.-   21. Takimoto j. 2009, Mol. Biosystems, 5, 931-34.-   22. Wang w. Nature Neuro. 2007, 8; 1063-1072.-   23. Ye, S. 2008, JBC 283, 1525-1533.-   24. Yanagisawa 2008 Chem & Biol. 15, 1187-1197.-   25. Wang et Al, 2011 Aijun Wang, Natalie Winblade Nairn, Marcello    Marelli and Kenneth Grabstein (2012). Protein Engineering with    Non-Natural Amino Acids, Protein Engineering, Prof. Pravin Kaumaya    (Ed.), ISBN: 978-953-51-0037-9, InTech, Available from:    http://www.intechopen.com/books/protein-engineering/protein-engineering-with-nonnatural-amino-acids-   26. Fekner, T., Li, X., & Chan, M. K. (2010). Pyrrolysine Analogs    for Translational Incorporation into Proteins. European Journal of    Organic Chemistry, 4171-4179.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

All patents and patent applications referred to herein are incorporatedby reference in their entirety.

1. A process for stabilizing a eukaryotic cell line which expressesPylRS and tRNAPyl and is capable of incorporating a gene encoding atarget protein comprising one or more non-natural amino acids encoded byan amber codon, wherein the process comprises culturing the cell line inthe presence of a decoy amino acid of formula VII:

wherein G=H, OH, —OCH₃, OCH₂CH₃, O—C(═O)—CH₃ or NH—K-Q; X=bond, CH₂, S,O, NH, N—(C═O)— or CH-J; J=alkyl, aryl, heteroaryl or the side chain ofone of the 20 natural amino acids; Y=bond, NH, O, S, CH₂; Z=O, NH, CH₂,S, CH—NH₂; K=CO or SO₂; a=0, 1, 2 or 3; b=0, 1, 2 or 3; Q=—H, C₁₋₆alkyl,aryl, heteroaryl —OC₁₋₆alkyl, —OCH₂aryl, —OCH₂heteroaryl, —C₂₋₆alkenylor —OC₂₋₆alkenyl; and R=C₁₋₆alkyl, C₂₋₆ alkenyl, —CH₂aryl, C₂₋₆alkynyl,C₁₋₆haloalkyl or C1-6azidoalkyl.
 2. A process for stabilizing aeukaryotic cell line which expresses PylRS and tRNAPyl and is capable ofincorporating a gene encoding a target protein comprising one or morenon-natural amino acids encoded by an amber codon, the processcomprising: (a) culturing the cell line in the presence of a targetprotein containing one or more non-natural amino acids encoded by anonsense codon; (b) culturing the cell line in the presence of a decoyprotein containing one or more non-natural amino acids encoded by anonsense codon, wherein the decoy protein is expressed under the controlof an inducible promoter; or (c) culturing the cell line whereinexpression of tRNAPyl occurs under the control of a repressiblepromoter.
 3. A process according to claim 2(b) wherein the decoy proteinis selected from Green fluorescence protein, Red Fluorescence Protein,Yellow Fluorescence Protein, Cyan Fluorescence Protein, bluefluorescence protein, albumin, SEAP, Actin, b-2 microglobulin,glutathione-s-transferase, IgG, and a poly amber containing peptide. 4.A stable eukaryotic cell line obtained by the process of claim
 2. 5. Astable eukaryotic cell line according to claim 4 which expresses PylRSand tRNAPyl and a decoy protein containing one or more non-natural aminoacids encoded by a nonsense codon, wherein the decoy protein isexpressed under the control of an inducible promoter.
 6. A process forproducing a stable eukaryotic cell line which is capable of expressingPylRS and tRNAPyl and incorporating a gene encoding a target proteincontaining one or more non-natural amino acids encoded by an ambercodon, the process comprising: (a) introducing into the cell line genesencoding PylRS and tRNAPyl and stably expressing PylRS and tRNAPyl inthe cell line; (b) culturing the resultant cell line in the presence of:(i) a decoy amino acid which is a substrate for PylRS but which isincapable of being incorporated into an extending protein chain; or (ii)a decoy protein expressed by the cell line wherein tRNApyl is expressedonly by the cell line at the same time as the decoy protein isexpressed.
 7. A process according to claim 6(i), further comprisingintroducing into the eukaryotic cell line a gene encoding a targetprotein containing one or more non-natural amino acids and expressingthe target protein in the absence of the decoy amino acid.
 8. A processaccording to claim 6(ii), wherein expression of the decoy protein isunder an inducible promoter systems including conditionally activatedpromoters and promoter systems such as the tetracycline regulatedpromoters (TetO or tTA; TetOn and TetOFF), doxycycline-inducible (TRE)promoters, cAMP inducible promoters, glucocorticoid activated promotersystems, IPTG inducible promoters (lac), Cd2+ or Zn2+ induciblepromoters (methalloprotein promoters), interferon dependent promoters(e.g. murine MX promoter), HIV LTR promoters (Tat), DMSO induciblepromoters (GLVP/TAXI, ecdysone), and rapamycin inducible promoters(CID).
 9. A process according to claim 6(ii), further comprisingintroducing into the eukaryotic cell line a gene encoding a targetprotein containing one or more non-natural amino acids such that thetarget protein is stably expressed in the cell line and expressing thetarget protein without expressing the decoy protein.
 10. A stableeukaryotic cell line obtained by a process according to claim
 6. 11. Aprocess for preparing a target protein containing one or morenon-natural amino acids encoded by an amber codon which comprisesintroducing into a stable eukaryotic cell line according to claim 10 agene encoding a target protein containing one or more non-natural aminoacids encoded by an nonsense codon such that the target protein isstably expressed in the cell line and expressing the target protein inthe presence of a source of the one or more non-natural amino acids and(i) in the absence of a decoy amino acid; or (ii) in the absence of aninducer of expression of a decoy protein.
 12. A process according toclaim 11 wherein the eukaryotic cell line is a mammalian cell line isselected from CHO, HEK293, PERC6, COS-1, COS-7, HeLa, VERO, mousehybridoma and mouse myeloma cell lines.
 13. A process according to claim11 which further comprises the step of chemically modifying theresultant target protein.